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| | <!-- Projectここから --> | | <!-- Projectここから --> |
| | <div id = "Project-block" style = "display:none"> | | <div id = "Project-block" style = "display:none"> |
| - | <!-- Project introduction -->
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| - | <div id = "Project-1">
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| - | <img src = "https://static.igem.org/mediawiki/2014/1/1c/Sub_introduction.png" class = "contTitle"/>
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| - | <p>The word "counter" may remind you of the machine with which you can count the number of objects, such as persons and vehicles. Some people familiar with electronic circuits may remind of the logic circuit. In each case, the system is regarded as memory device that remember the number of inputs, which is important for our lives.</p>
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| - | <p>In the natural world, cellular counters also memorize the number of events. For example, there are telomere length regulation[1][2], cell aggregation[3][4], etc. Telomere length of <i>Saccharomyces cerevisiae</i> is regulated by the number of the Rap1 protein, indicating the existence of counting system. The cell aggregation size of <i>Dictyostelium</i> is regulated by counting factor (CF). CF counts the number of aggregating cells and negatively regulates the cell adhension. In these ways, cellular counters are widely utilized for the regulation of biological systems.</p>
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| - | <p>With synthetic biological approach, Ari et al constructed a cellular counter termed the riboregulated transcriptional cascade (RTC) counter[5]. The state transition occurs after an arabinose induction (Fig. 1). The system is regulated by riboregulators. The Biobrick part of this cellular counter has already existed, which was constructed by Tokyo-Nokogen 2009 and was named BBa_K225002, BBa_K25003 [6]. </p>
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| - | <p>In order to expand the function of this counter, we added "reset system". The reset system enables the transition from any state to the initial state after a particular input. The property is expected to apply for a deterministic finite automaton, which is the system developed from information science. Within the finite number of states, the system makes the transition to another state in responce to a particular input.</p>
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| - | <p>As the key of its resetting mechanism, our counter utilizes the regulation system based on sigma factor and anti-sigma factor. Sigma factor is a subunit of RNA polymerase and help it bind to the specfic sequence of the promoter. Anti-sigma factor blocks sigma factors from interacting with RNA polymerase. The utilization of this regulation system brings about benefit for attempt to make automaton.Firstly, the number of states can be increased easily because what we have to concern is the combination of sigma and anti-sigma factor. Secondly, the crosstalk between sigma and anti-sigma factors can be circumvented even if you raise the number of states. That is because we can choose such combinations between sigma and anti-sigma factors that have little crosstalk.</p>
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| - | <img src = "https://static.igem.org/mediawiki/2014/b/b2/Irie_induce.png" class = "figure" />
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| - | <p>Fig. 1 The concept of RTC counter. After first, second and third induction of arabinose, the state of cells moves from 0 to 1, 1 to 2 and 2 to 3, respectively.</p>
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| - | </div>
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| - | <!-- Project σ-memory -->
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| - | <div id = "Project-2">
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| - | <img src = "https://static.igem.org/mediawiki/2014/e/e4/Sub_memory.png" class = "contTitle" />
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| - | <h3>sigma factor</h3>
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| - | <p class = "noIndent">Sigma factor is a subunit of RNA polymerase related to promoter recognitions. There are two types of sigma factors, one that is housekeeping (expressed constantly) and another that is expressed under some conditions. It is possible to deliver genes of sigma factors that are not derived from some species to another species, so we can use many kinds of sigma factor. Which promoter RNA polymerase tends to bind to is decided by which sigma factor RNA polymerase is bound. Especially some sigma factors promote only transcription of a specific promoters. Therefore if we choose a set of sigma factors and promoters skillfully, we can controll transcription without crosstalk. Here, “without crosstalk” means every sigma factor in the set promotes only transcription of the cognate promoter.</p>
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| - | <img src = "https://static.igem.org/mediawiki/2014/2/2f/Nakashima_image%281-0%29.png" class = "figure-height" />
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| - | <p>Anti-sigma is also a protein which is related to transcriptional control by sigma factors. Anti-sigma prevents sigma factors from binding to RNA polymerase. Consequently, sigma factor cannot activates transcription of cognate promoters. Similarly as sigma factor, there are many kinds of anti-sigma and some anti sigmas prevent only specific sigma factors from transcriptional control. Therefore if we choose a set of sigma factors, anti sigmas, and promoters skillfully, we can activate control, not only activate but also repress, transcription without crosstalk. </p>
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| - | <img src = "https://static.igem.org/mediawiki/2014/a/a2/Nakashima_image%281-1%29.png" class = "figure-height" />
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| - | <p>Transferred sigma factors or anti sigmas may have negative effects on the growth of the host cell. For example, anti sigma may prevent housekeeping sigma factors from transcriptional control. However it is no problem since not all sigma factors and anti sigmas have negative effect and there are many kind of sigma factors and anti sigmas.［1］</p>
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| - | <h3>sigma-memory construction</h3>
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| - | <img src = "https://static.igem.org/mediawiki/2014/f/f8/Nakashima_iroiro.png" class = "figure" />
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| - | <p class = "noIndent">This is the construction of our sigma-memory.This gene circuits is composed of three parts. The gene of a sigma factor is placed at the downstream of the promoter that is induced by a substance A and at the downstream of Psigma, which is induced by the sigma factor. The gene of the cognate anti sigma is placed at the downstream of promoter which is induced by a substance B.</p>
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| - | <p>At first, there is no sigma factors. If input A exists, sigma factor is expressed. Then the positive feedback circuits of sigma factor starts producing sigma factor, and consequently sigma factor will remain. Though if input B exists, anti-sigma is expressed and the positive feedback circuits is inhibited. Both sigma factor and anti-sigma are subjects to degradation［2］, so all of them are decomposed after some time and sigma-memory returns to its original state.</p>
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| - | <img src = "https://static.igem.org/mediawiki/2014/a/af/Nakashima_induced.png" class = "figure" />
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| - | <p>We can regard existence/absence of sigma factor as 1/0 of memory, and this values of memory can switch by input A or input B. Using the promoter which is cognate to the sigma factor, the information whether the value of memory is 1 or 0 can be derived. For example, consider the circuits on the right. The repotor is expressed if and only if sigma-memory's value is 1 (i.e. sigma factor exists).</p>
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| - | <img src = "https://static.igem.org/mediawiki/2014/8/8f/Nakashima_sigma.png" class = "figure" />
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| - | <img src = "https://static.igem.org/mediawiki/2014/e/ef/Nakashima_reset_func01.png" class = "figure" style="width:400px;float:left;"/>
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| - | <img src = "https://static.igem.org/mediawiki/2014/5/5a/Nakashima_reset_func02.png" class = "figure" style="width:400px;float:left;" />
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| - | <p style="clear:both">In addition, no crosstalk sets of sigma factors, promoters, and anti-sigmas enable us to make multi-sigma-memory gene circuits. To make the explanation easier, consider the case in which E. coli has two sigma memories, sigmaA-memory and sigmaB-memory. SigmaA The value of sigmaA-memory change from 0 to 1 if input A1 exists and change from 1 to 0 if inputs B1 exists. Also the value of sigmaB-memory change from 0 to 1 if input A2 exists and change from 1 to 0 if input B2 exists. If the four input A1, B1, A2, and B2 have no crosstalk, sigmaA-memory and sigmaB-memory also have no crosstalk. For example, when input A1 exists, only the value of sigmaA-memory change from 0 to 1 since sigmaA promotes only transcription from PsigmaA (promoter that is cognate to sigmaA). Since the transcription from PsigmaB is not activated, sigmaB is not expressed and the value of sigmaB dose not change. The same is true of input A2. When input B1 exist, anti sigmaA is expresses and the value of sigmaA-memory changes from 1 to 0. However, anti sigmaA has no effect on the transcription of PsigmaB and the value of sigmaB dose not change. The same is also true for the input B2. So it can be confidently said that E. coli has two sigma memories.</p>
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| - | <img src = "https://static.igem.org/mediawiki/2014/6/6f/Nakashima_sigma_memory.png" class = "figure" />
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| - | <p>Since this construction has a positive feedback, leakage of promoter may be a serious problem. If the promoter has leakage and sigma factor is expressed when input A dose not exist, this error may be enlarged by positive feedback. (Whether the error is really enlarged depends on whether the leakage of sigma factor is large compared to the degradation of sigma factor.) However if the leakage of anti-sigma is considerably large, sigma factor dose not produced from positive feedback when leak of sigma factor occurs. Therefore leakage is not an obstacle of our project if we choose the promoter skillfully.</p>
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| - | <h3>sigma-memory and automaton</h3>
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| - | <p class = "noIndent">Speaking easily, an automaton is referred to what has states and transition rules. Every state has a transition rule, say it is decided what is the next state if an input exists. For example, the automaton on the right has five states, 0, 1, 2, 3, 4, and transition rules are represented by arrows. If the current state is 0 and the input is b, the next state is 2. Transition rules having loops or feedbacks are allowed.</p>
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| - | <p>sigma-memory can be considered an automaton. Every tuple of the value if sigma-memory (say, (1, 1, 0, 1, 0)) corresponds to a state, and the promoter which is used in the construction of sigma-memory decides transition rules. It is possible to use the value of one sigma-memory as an inputs of another sigma-memory. This fact enables us to make complicated transition rules.</p>
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| - | <p>For example, consider the case where E. coli has two sigma-memory. The value of sigmaA-memory( a sigma-memory which uses sigmaA in the construction as a sigma factor) change from 0 to 1 if a substance A1 exists, also the value of sigmaB-memory change from 0 to 1 if the value of sigmaA and a substance A2 exists. (This condition can be written in terms of mathematical logic, namely AND(sigmaA, IPTG)) Gene circuits which work as logic circuits (says AND, OR, NAND, etc...) have been designed.［3］ Therefore such gene circuit can be made. The value of sigmaA-memory changes from 1 to 0 when a substance B1 exists and that of sigmaB-memory changes from 1 to 0 when a substance B2 exist. In this case, it is characteristic that sigmaB-memory is affected by sigmaA memory. These gene circuits can be considered as the automaton on the below. </p>
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| - | <img src ="https://static.igem.org/mediawiki/2014/8/8f/Nakashima_diagram.png" class = "figure" />
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| - | </div>
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| - | <!-- Project Construction -->
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| - | <div id = "Project-3">
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| - | <img src = "https://static.igem.org/mediawiki/2014/b/b9/Sub_construction_of_counter.png" class = "contTitle" />
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| - | <h3>resettable counter by sigma-memory</h3>
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| - | <p class = "noIndent">Resettable counter is a device that can count the number of induction events of arabinose, and expresses a reporter correspond to each states. In addition, the count can be reset by IPTG induction. Resettable counter can be considered as an automaton. The automaton has two inputs, input A and input B. Each states corresponds to the count, namely the states of the automaton is 0,1,2,...etc. The transition rule is very simple. When the current states is n and the input is A, the next states is n+1, and when the current state is n and the input is B, the next states is 0. Since a counter is one kind of automata, sigma-memory can be applied for making resettable counters. </p>
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| - | <img src = "https://static.igem.org/mediawiki/2014/3/35/Nakashima_012.png" class = "figure" />
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| - | <p>2-counter is a counter that can count up to 2, and is an automaton that has three states, 0, 1, and 2. This automaton can be constructed by sigma-memory as following. Two kinds of sigma factor are necessary for constructing the 2-counter, so for the convenience we will call these two kinds of sigma factors sigmaA and sigmaB. In state 0, both sigmaA and sigmaB do not exist (i.e. sigmaA-memory and sigmaB-memory is 0). In state 1, only sigmaA exists and in states 2, both sigma factors exist. The input is the induction of arabinose or IPTG, and the transition rules with it are showed in the Figure.</p>
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| - | <p>A gene circuit that realizes this automaton can be represented as following. The value of sigmaA changes from 0 to 1 after arabinose induction (i.e. sigmaA is expressed when arabinose exists). This change corresponds to the transition from state from 0 to 1.The value of sigmaB-memory changes from 0 to 1 when both arabinose and sigmaA exist. The conditional branch can be made by using AND function. This change corresponds to the transition from state 1 to state 2. Both the value of sigmaA-memory and sigmaB-memory change from 1 to 0 after IPTG induction (i.e. anti-sigmaA and sigmaB is expressed when IPTG exist).</p>
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| - | <h3>riboregulator</h3>
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| - | <p class = "noIndent">According to the discussion in 2-1, it is necessary to use AND function for the construction of a resettable counter. Therefore a riboregulator is used for the realization of the AND function.</p>
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| - | <img src = "https://static.igem.org/mediawiki/2014/f/f2/Nakashima_cr_structure.png" class = "figure" />
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| - | <p>A riboregulator is a post-transcriptional regulation system composed of two kinds of RNAs, Cis-repressed mRNA (crRNA) and trans-activating RNA (taRNA). CrRNA forms a stem-loop and its ribosomal binding site (RBS) is covered. Consequently the gene coded in cis-repressed mRNA isn't translated. However if trans-activating RNA exist, crRNA and taRNA are hybridized and RBS gets exposed and translation starts.［4］</p>
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| - | <p>Namely, the gene coded in crRNA is expressed only when both crRNA and taRNA is transcripted. Therefore this system can be considered as the AND function. For example, consider the gene circuit, Plac-taRNA-d.term-Pbad-cr-RBS-GFP-d.term.(cr is the sequence in crRNA which binds to RBS) If and only if both IPTG and arabinose exists, GFP is expressed. Therefore, the riboregulator can combine two promoters and produce AND functions</p>
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| - | <h3>resettable counter construction</h3>
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| - | <img src = "https://static.igem.org/mediawiki/2014/8/81/Nakashima_const.png" class = "figure" />
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| - | <p class = "noIndent">The construction of our resettable counter is explained here.</p>
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| - | <p>Ecf20_992 (Sigma20) and Ecf11_3726 (Sigma11) are used as sigmaA and sigmaB as mentioned above respectively. These two sigma factors strongly activate the cognate promoters, and their inhibition of growth is negligible. ［1］The promoters correspond to sigma20 and sigma11 are Pecf20_992(Psigma20) and Pecf11_3726(Psigma11), respectively. The sequences of these two promoters has no restriction site of Ecor1, Xba1, Spe1, and Pst1. The cognate anti-sigmas are anti-20 and anti-11 respectively. These two anti-sigmas considerably prevent the cognate sigma factors from activating transcription, and their inhibition of growth is also negligible. These sigma factors, cognate promoters, and cognate anti-sigmas has no cross talk.</p>
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| - | <p>Cis-repress sequence is crR12 and trans-activating RNA is taR12. This pair is selected because the leakage is small. ［4］The reporter in this construction is GFP.</p>
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| - | <h3>mechanism and extension</h3>
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| - | <p class = "noIndent">At first, both the value of sigma20-memory and sigma11-memory are 0. Only the crRNA coding sigma20, which is at downstream of constitutive promoter is translated. After the first induction of arabinose, taRNA at the downstream of PBAD is transcribed and the crRNA coding sigma20 is translated, and the value of sigma20-memory changes from 0 to 1. Since sigma20 exists, crRNA coding sigma11 at the downstream of Psigma20 is transcribed. After the second induction of arabinose, taRNA is transcribed and sigma11 is expressed, and the value of sigma11-memory changes from 0 to 1. At this time, GFP at the downstream of Psigma11 is expressed and we can check the count as 2.</p>
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| - | <p>After an induction of IPTG, anti-sigma20 and sigma11 are expressed, and the value of sigma20-memory and sigma11-memory will be 0. Therefore the count is reset.</p>
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| - | <p>Since there are many kinds of pair of sigma factors and cognate promoters which have no crosstalk, n-counter can be made by the same way. Besides, the construction of 2-counter can be simplified.</p>
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| - | <img src = "https://static.igem.org/mediawiki/2014/4/41/Nakashima_sigmaconst.png" class = "figure" />
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| - | <p>This simplified counter is made by only one sigma factor. It works as the same way as original 2-counter unill 1 count. After 1 count, crRNA coding GFP is transcribed at the downstream of Psigma. Therefore when the next arabinose induction occurs, GFP is translated. This expression can be considered a report of 2 count. This simplified counter can be also extended to n count. Simplified counter can count up to larger numbers compared to the original counter even when the same number of sigma factors are used, but cannot be reset from their final count. We did experiments on this simplified counter.</p>
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| - | <h3>comparison with previous counter</h3>
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| - | <p class = "noIndent">Our sigma-Recounter is improved version of the previous counter constructed by Ari.［5］In the previous counter, T7 RNA polymerase and T3 RNA polymerase is used, while in our counter sigma factors is used. Using sigma factor has two merits. One is the ease of extension for n-counter. The number of RNA polymerase derived from virus is limited, but sigma factor has great divergence. Consequently it is more easy to construct n-counter by using sigma factor. Another is the existence of inhibitor. Anti-sigma is inhibitor of sigma factor which has no cross talk. Inhibitor is necessary to realize reset function.</p>
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| - | <p>Other difference is the positive feedback circuits. Previous counter has no feedback circuits. Since sigma factor is more subject to degradation than RNA polymerase, positive feedback circuits is necessary to keep "memory" (i.e. for sigma factor to remain) in our counter.</p>
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| - | </div>
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| - | <!-- Project application -->
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| - | <div id = "Project-4">
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| - | <img src = "https://static.igem.org/mediawiki/2014/a/af/Sub_application.png" class = "contTitle" />
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| - | <p> As described above, the genetic circuit we constructed can be considered as an automaton. In a previous study[1], many sigma and anti-sigma that regulate transcription without crosstalk have been reported. Thus, an automaton that has many states can be constructed. Furthermore, though in this project reset is transition from other states to state 0, more general system that is capable of changing one state to any other states is possible. Thus more general automaton by genetic circuits may be possible. Examples of an automaton are such as biocomputer or lifegame etc. Bringing this concept from algorithmic world into synthetic biology is our challenge. Though in our project input is a single short intermittent signal, a more general circuit that responds to more general input is possible to be considered by integrating additional circuits into our circuit.</p>
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| - | <p> For example, the change of balance between 2 substances itself can be considered as a input. Considering substances A and B, this additional circuit is possible:</p>
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| - | <p>pA-repressorB-reporterA-activatorX-pX-repressorA</p>
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| - | <p>pB-repressorA-reporterB-activatorY-pY-repressorB</p>
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| - | <img src ="https://static.igem.org/mediawiki/2014/b/ba/Sumi_AB.png" class = "figure" />
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| - | <p> Here, substances A/B activate promter A/B. This additional circuit essentially contains toggle switch structure with delay negative feedback loop. For example, when substance A become dominant against substance B, the toggle switch amplifies the dominance of promoter A and the following negative feedback suppress the dominance. Consequently, this additional circuit is expected to convert the change of the dominance to a pulse expression of reporter protein. Therefore, this additional circuit can expand the range of input. As this circuit can be a monitor of a milieu if A is industrial waste, our genetic circuit can be applied to much wider range of problems.</p>
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| - | </div>
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| | </div> | | </div> |
| | <!-- Result --> | | <!-- Result --> |
| | <!-- Lab Data --> | | <!-- Lab Data --> |
| | <div id = "Lab-block" style = "display:none;"> | | <div id = "Lab-block" style = "display:none;"> |
| - | <div id = "Lab-1">
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| - | </div>
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| - | <div id = "Lab-2">
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| - | </div>
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| - | <div id = "Lab-3">
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| - | <h1>lab note</h1><br>
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| - | <h3>7,14,2014</h3><br>
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| - | <br>
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| - | <h3>Lab member</h3> Yoshikawa,Nakashima<br>
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| - | <br>
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| - | <h3>Contents</h3><br>
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| - | <h5>Making Plate Culture</h5><br>
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| - | Ampicillin *10<br>
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| - | Chloramphenicol*6<br>
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| - |
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| - | <h3>7,15,2014</h3><br>
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| - | <h3>Lab member</h3>Yoshikawa,Nakashima<br>
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| - | <br>
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| - | <h3>Contents</h3><br>
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| - | <h5>Making DW 100mL</h5><br>
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| - | <br>
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| - | <h5>TF</h5><br>
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| - | 4-17F(A-1), 4-4E(A-2), 3-19O(A-3), 3-4G(A-4), 4-1N(A-6), 3-3F(A-8), 4-13L(B-10)<br>
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| - | <br>
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| - | <h3>7,16,2014</h3><br>
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| - | <h3>Lab member</h3>Yoshikawa,Nakashima<br>
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| - | <br>
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| - | <h3>Contents</h3><br>
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| - | A-2:No colony<br>
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| - | <h5>TF</h5>A-2(recovery)<br>
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| - | <h5>preculture</h5>A-1, A-3, A-4, A-6, A-8, B-10,<i>Escherichia coli</i> JM109(for competent cell)<br>
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| - | <br>
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| - | <h3>7,17,2014</h3><br>
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| - | <h3>Lab member</h3>Yoshikawa,Nakashima<br>
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| - | <br>
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| - | <h3>Contents</h3><br>
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| - | A-2:There are something like colonies.<br>
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| - | Making glycerol stock/<h5>miniprep</h5><br>
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| - | A-1, A-3, A-4, A-6, A-8<br>
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| - | All samples:consentration is low.<br>
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| - | B-10:disposed<br>
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| - | <br>
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| - | <h5>making reagent</h5><br>
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| - | LB: 500mL<br>
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| - | 50mM CaCl2: 400mL<br>
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| - | 50mM CaCl2/ 20% glycerol: 200mL<br>
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| - | <br>
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| - | <h5>preculture</h5><br>
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| - |
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| - | A-1 #1<br>
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| - | A-2 #1 #2<br>
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| - | A-3 #1<br>
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| - | A-4 #1 <br>
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| - | A-6 #1<br>
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| - | A-8 #1<br>
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| - | B-10 #1 #2<br>
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| - | <i>E. coli</i> JM109（for competent cell）<br>
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| - | <br>
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| - | #:colony number<br>
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| - | <br>
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| - | <h3>7,18,2014</h3><br>
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| - | <h3>Lab member</h3>Yoshikawa,Nakashima<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>miniprep</h5><br>
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| - | A-1,A-3,A-6:from culture 2.5ml OK<br>
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| - | A-4,A-8:from culture 1.5ml OK<br>
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| - | A-2#1:made glycerol stock. Low consentration.<br>
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| - | B-10#1:Low consentration.<br>
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| - | A-2#2,B-10#2:did not grow<br>
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| - | <br>
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| - | <h5>Making competent cell</h5><br>
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| - | 100uL*120<br>
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| - | <br>
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| - | <h5>making reagent</h5><br>
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| - | （2000×）Ampicillin 3ｍL （100mg/mL）<br>
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| - | <br>
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| - | <h3>7,22,2014</h3><br>
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| - | <h3>Lab member</h3>Yoshikawa,Nakashima<br>
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| - | <br>
| |
| - | <h3>Contents</h3><br>
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| - | <h5>Cut check</h5><br>
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| - | A-4 (81ng/μL):ES Cut,XP cut.Checked in 1.5h, 2h, 2.5h, 3h:OK<br>
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| - | Left:1kbp Ladder/A-4(Control)/ES 1.5h/ES 2h/ES 2.5h/ES 3h<br>
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| - | Right:1kbp Ladder/A-4(Control)/XP 1.5h/XP 2h/XP 2.5h/XP 3h<br>
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| - | (ここに写真20140722_1を貼付）<br>
| |
| - | <br>
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| - | <h5>TF</h5><br>
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| - | We measured cfu of competent cell we made.<br>
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| - | We used Efficiency Kit （RFP Construct on pSB1C3）in distribution Kit.<br>
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| - | <h5>TF</h5> : 50pg, 20pg, 10pg, 5pg<br>
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| - | competent cell:25uL<br>
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| - | Sprinkling:100uL<br>
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| - | <br>
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| - | <h5>preculture</h5><br>
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| - | B-10 #3, A-4<br>
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| - | <br>
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| - | <h3>7,23,2014</h3><br>
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| - | <h3>Lab member</h3>Yoshikawa,Nakashima<br>
| |
| - | <br>
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| - | <h3>Contents</h3><br>
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| - | plate check:Did not grow.<br>
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| - | <br>
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| - | <h5>miniprep</h5><br>
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| - | A-4, B-10 #3(made glycerol stock):OK<br>
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| - | <br>
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| - | Making plate<br>
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| - | 200ml,CP*10<br>
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| - | <br>
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| - | <h3>7,28,2014</h3>
| |
| - | <h3>Lab member</h3>Nakashima<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>digestion,gel extraction</h5><br>
| |
| - | A-6 SP, B-10 XP：disposed<br>
| |
| - | 100bp Ladder, A-6 SP, B-10 XP, NC<br>
| |
| - | (ここに20140728_1を貼付）<br>
| |
| - | (wrote at 0731<br>
| |
| - | A-6<br>
| |
| - | band near 2kbp:SP cut or single cut<br>
| |
| - | band near 1.5kbp:Plasmid)<br>
| |
| - | <br>
| |
| - | <h5>colony PCR</h5><br>
| |
| - | A-6 #1, B-10 #1<br>
| |
| - | 1kbp Ladder, A-6, B-10, NC<br>
| |
| - | (ここに20140728_2を貼付)<br>
| |
| - | A-6:OK,B-10:wrong<br>
| |
| - | <br>
| |
| - | <h5>Measuring cfu</h5><br>
| |
| - | competent cell:50uL<br>
| |
| - | <br>
| |
| - | <h3>7,29,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa,Nakashima<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | Plate check:All Plates did not grow.<br>
| |
| - | <br>
| |
| - | <h5>TF</h5><br>
| |
| - | B-10 by electroporation.<br>
| |
| - | <br>
| |
| - | <h5>Measuring cfu</h5><br>
| |
| - | A-1:13,65,130pg<br>
| |
| - | <br>
| |
| - | <h5>Electrophoresis check</h5><br>
| |
| - | 1kbp Ladder, A-6(Plasmid), NC<br>
| |
| - | (ここに20140729_1を貼付）<br>
| |
| - | There is band in 1500kbp,so 1500kbp in 0728 may be rest of cutting.<br>
| |
| - | No contamination in Dye.<br>
| |
| - | <br>
| |
| - | <h3>7,30,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa,Nakashima<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>colony check</h5><br>
| |
| - | cfu:Did not grow<br>
| |
| - | B-10:One colony<br>
| |
| - | <br>
| |
| - | <h5>Cut check</h5><br>
| |
| - | We checked whether A-6 band in 0728 was rest of cutting.<br>
| |
| - | A-1 SP cut:checked on different times.<br>
| |
| - | <br>
| |
| - | <h5>colony PCR</h5><br>
| |
| - | B-10(from Plate made in 0729)<br>
| |
| - | <br>
| |
| - | 1kbp Ladder, 1.5h, 2h, 2.5h, B-10(colony PCR), NC<br>
| |
| - | （ここに20140730_1を貼付）<br>
| |
| - | band near 800bp:<i>RFP</i> between <i>Spe1</i> site and <i>Pst1</i> site of A-1.<br>
| |
| - | band near 2kbp:Backbone cut by SP cut<br>
| |
| - | band near 3kbp:rest of cutting<br>
| |
| - | <br>
| |
| - | 1.5h:incompletely cut<br>
| |
| - | 2h,2.5h:OK<br>
| |
| - | We decided 2h for digestion.<br>
| |
| - | B-10:OK<br>
| |
| - | <br>
| |
| - | <h5>preculture</h5><br>
| |
| - | B-10<br>
| |
| - | <br>
| |
| - | <h3>7,31,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa,Nakashima<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>miniprep</h5><br>
| |
| - | B-10 #1（from 140729 Plate）(made glycerol stock)<br>
| |
| - | <br>
| |
| - | <h5>digestion,gel extraction</h5><br>
| |
| - | A-6 SP, B-10 XP<br>
| |
| - | <br>
| |
| - | 1kbp Ladder, A-6, B-10<br>
| |
| - | （ここに20140731_1を貼付）<br>
| |
| - | B-10:incomplete cut<br>
| |
| - | others:OK<br>
| |
| - | <br>
| |
| - | <h5>ligation</h5><br>
| |
| - | C-7 (A-6 SP + B-10 XP)<br>
| |
| - | <br>
| |
| - | <h3>8,1,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa,Nakashima<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>TF</h5><br>
| |
| - | C-7（Chemical & EP）<br>
| |
| - | <br>
| |
| - | <h5>ligation Check</h5><br>
| |
| - | PCR reaction primed with Universal Primer, using ligation products as template.<br>
| |
| - | （ここに20140801_1を貼付）<br>
| |
| - | OK!<br>
| |
| - | <br>
| |
| - | <h3>8,4,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa,Nakashima<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>Plate check</h5><br>
| |
| - | No colony<br>
| |
| - | <br>
| |
| - | <h5>digestion</h5><br>
| |
| - | A-6 SP, B-10 XP<br>
| |
| - | <br>
| |
| - | <h5>Electrophoresis</h5><br>
| |
| - | 1kbp Ladder, A-6, B-10<br>
| |
| - | (ここに20140804_1を貼付）<br>
| |
| - | Incomplete cut.Disposed.<br>
| |
| - | <h5>preculture</h5><br>
| |
| - | B-10<br>
| |
| - | <br>
| |
| - | <h3>8,5,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa,Nakashima<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>miniprep</h5><br>
| |
| - | B-10 →Sample Lost! Bye bye, <i>GFP</i>!<br>
| |
| - | <br>
| |
| - | <h5>Cut check</h5><br>
| |
| - | SP(A-1), XP(A-1), EX(A-4), ES(A-4)<br>
| |
| - | 2h, 2.5h, 3h <br>
| |
| - | <br>
| |
| - | <h5>Elrctrophoresis</h5><br>
| |
| - | 1kbp Ladder, A-1, A-1 SP(2h, 2.5h, 3h), A-1 XP(2h,2.5h, 3h), A-4,<br>
| |
| - | A-4 EX(2h, 2.5h 3h), A-4 ES(2h, 2.5h, 3h), None, 1kbp Ladder<br>
| |
| - | （ここに20140805_1を貼付）<br>
| |
| - | Incomplete cut.<br>
| |
| - | <br>
| |
| - | <h5>preculture</h5><br>
| |
| - | A-1, A-4, B-10<br>
| |
| - | <br>
| |
| - | <h3>8,6,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>miniprep</h5><br>
| |
| - | A-1, A-4, B-10<br>
| |
| - | <br>
| |
| - | <h5>digestion, gel extraction</h5><br>
| |
| - | A-6 SP, B-10 XP<br>
| |
| - | <br>
| |
| - | 1kbp Ladder, A-6, B-10
| |
| - | (ここに20140806_1を貼付）<br>
| |
| - | A-6:OK<br>
| |
| - | B-10:Incomplete cut<br>
| |
| - | (ここに20140806_2を貼付）<br>
| |
| - | <br>
| |
| - | We chenged restriction enzyme.<br>
| |
| - | B-10 XP<br>
| |
| - | (ここに20140806_3を貼付）<br>
| |
| - | OK!<br>
| |
| - | （ここに20140806_4を貼付）<br>
| |
| - | <br>
| |
| - | <h5>making gel</h5>200ml<br>
| |
| - | <br>
| |
| - | <h5>ligation</h5><br>
| |
| - | C-7（A-6 SP + B-10 XP）<br>
| |
| - | <br>
| |
| - | <h5>TF</h5><br>
| |
| - | 4-11L, C-7<br>
| |
| - | <br>
| |
| - | <h3>8，7,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa,Nakashima,Tara,Itoh,Tsukada<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>colony PCR</h5><br>
| |
| - | C-7<br>
| |
| - | (ここに20140807_1を貼付）<br>
| |
| - | (ここに20140807_2を貼付）<br>
| |
| - | All bands are self ligation of backbone.<br>
| |
| - | <br>
| |
| - | <h5>digestion, gel extraction</h5><br>
| |
| - | A-6 SP, B-10 XP<br>
| |
| - | <br>
| |
| - | 1kbp Ladder, B-10, A-6<br>
| |
| - | (ここに20140807_3を貼付）<br>
| |
| - | <h5>ligation</h5><br>
| |
| - | C-7 (A-6 SP + B-10 XP)<br>
| |
| - | <br>
| |
| - | <h5>Cut check</h5> (O/N)<br>
| |
| - | （enzyme-buffer)
| |
| - | S-B, S-H*, S-M*, P*-FD, E-FD<br>
| |
| - | *:Takara's product<br>
| |
| - | <br>
| |
| - | <h3>8,8,2014</h3><br>
| |
| - | <h3>Lab member</h3>Nakashima,Nakamura,Yamanaka,Yoshikawa(Fresh),Yoshikawa<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>Cut check sample Electrophoresis</h5><br>
| |
| - | <br>
| |
| - | 1kbp Ladder, S-B, S-H*, S-M*, NC, E-FD, P*-FD<br>
| |
| - | (ここに20140808_1を貼付）<br>
| |
| - | FD buffer is appropriate for SP cut.<br>
| |
| - | <br>
| |
| - | <h5>digestion, gel extraction</h5><br>
| |
| - | A-6 SP, B-10 XP<br>
| |
| - | (ここに20140808_2を貼付）<br>
| |
| - | (ここに20140808_3を貼付）<br>
| |
| - | OK!<br>
| |
| - | <br>
| |
| - | <h5>ligation</h5><br>
| |
| - | C-7 (A-6 SP + B-10 XP)<br>
| |
| - | <br>
| |
| - | <h5>TF</h5><br>
| |
| - | C-7,4-11L(culture in test tube) <br>
| |
| - | <br>
| |
| - | <h5>overlap extension PCR →PCR clean up</h5><br>
| |
| - | 1+2<br>
| |
| - | anealing 57℃,extension 10 sec, 30 cycle.<br>
| |
| - | <br>
| |
| - | 100bp Ladder, Pecf11, Pecf20, crRBS, taRNA<br>
| |
| - | (ここに20140808_4を貼付)<br>
| |
| - | OK!(low concentration)<br>
| |
| - | <br>
| |
| - | <h5>making gel</h5>200ml<br>
| |
| - | <br>
| |
| - | <h3>8,9,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>Plate and test tube check</h5><br>
| |
| - | 4-11L:OK!mixed with glycerol, and conserved in -80℃.<br>
| |
| - | C-7:OK! conserved in refrigerator.<br>
| |
| - | <br>
| |
| - | <h3>8,11,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa,Nakashima,Yamanaka,Nakamura<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>colony PCR</h5><br>
| |
| - | C-7 #1~11<br>
| |
| - | #1~4, NC<br>
| |
| - | （ここに20140811_1を貼付）<br>
| |
| - | #5~11,NC<br>
| |
| - | (ここに20140811_2を貼付)<br>
| |
| - | #10 is OK!<br>
| |
| - | <br>
| |
| - | <h5>overlap extension PCR →PCR clean up</h5><br>
| |
| - | Pecf11, Pecf20, crRBS, taRNA
| |
| - | (1+2)+3,PCR product<br>
| |
| - | 1kbp Ladder, Pecf11(1+2+3), Pecf20(1+2+3), crRBS(1+2+3)<br>
| |
| - | taRNA(1+2+3), Pecf11(all), Pecf20(all), crRBS(all), taRNA(all)<br>
| |
| - | (ここに20140811_3を貼付）<br>
| |
| - | <br>
| |
| - | <h5>Making Plate</h5><br>
| |
| - | Ampicillin*20<br>
| |
| - | <br>
| |
| - | <h5>preculture</h5><br>
| |
| - | A-6, B-10, C-7 #10<br>
| |
| - | <br>
| |
| - | <h3>8,12,2014</h3><br>
| |
| - | <h3>Lab member</h3>Nakashima,Nakamura,Yamanaka,Yoshikawa(Fresh),Yoshikawa<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>miniprep</h5><br>
| |
| - | A-6, B-10, C-6 #10 (made glycerol stock)<br>
| |
| - | <br>
| |
| - | <h5>digestion →gel extraction</h5><br>
| |
| - | A-8 EX, C-6 ES, pSB1A2(A-1) XP *2 sample, A-9 linear XP<br>
| |
| - | A-10 linear XP, A-7 linear XP, B-1 linear XP<br>
| |
| - | <br>
| |
| - | 1kbp Ladder, A-1 XP, A-1 XP, A-8 EX, C-6 ES<br>
| |
| - | (ここに20140812_1を貼付）<br>
| |
| - | C-6:wrong<br>
| |
| - | A-1:OK!<br>
| |
| - | <br>
| |
| - | <h5>colony PCR</h5><br>
| |
| - | 4-11L<br>
| |
| - | (ここに20140812_2を貼付）<br>
| |
| - | OK!<br>
| |
| - | <h5>ligation →TF</h5><br>
| |
| - | A-9, A-10, A-7, B-1<br>
| |
| - | <br>
| |
| - | <h5>Making reagent</h5><br>
| |
| - | LB 800ml<br>
| |
| - | <br>
| |
| - | <h3>8,13,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa,Nakashima,Tara,Nakamura,Takemura,Tsukada<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>miniprep</h5><br>
| |
| - | 4-11L（made glycerol stock）<br>
| |
| - | <br>
| |
| - | <h5>digestion→ gel extrction</h5><br>
| |
| - | A-8 EX, C-6 ES, A-6 SP, 4-11L XP<br>
| |
| - | <br>
| |
| - | 1kbp Ladder, A-6 SP, A-8 EX, C-6 ES, 4-11L XP<br>
| |
| - | (ここに20140813_1貼付）<br>
| |
| - | (ここに20140813_2貼付)<br>
| |
| - | C-6:Incomplete cut<br>
| |
| - | Backbone of 4-11L said to be pSB1A2, but ,actually,may be pSB1AK3.<br>
| |
| - | <br>
| |
| - | <h5>ligation→TF</h5><br>
| |
| - | D-7 (4-11L XP + A-6 SP), D-7(C-6 ES + A-8 EX)<br>
| |
| - | <br>
| |
| - | <h5>colony PCR</h5><br>
| |
| - | A-7 #1~2, A-9 #1~4, A-10 #1~4, B-1 #1~2 ,100bp Ladder<br>
| |
| - | (ここに20140813_3を貼付)<br>
| |
| - | A-7 #1, A-10 #4：OK!<br>
| |
| - | <br>
| |
| - | <h5>preculture</h5><br>
| |
| - | A-8, C-6, A-7 #1, A-10 #4<br>
| |
| - | <br>
| |
| - | <h3>8,14,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa,Nakashima,Tara,Takemura,Nakamura<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>miniprep</h5><br>
| |
| - | A-7 #1（made glycerol stock）, A-10 #4（made glycerol stock）, A-8, C-6<br>
| |
| - | <br>
| |
| - | <h5>overlap extension PCR</h5><br>
| |
| - | Pecf 11, Pecf 20, crRBS, taRNA<br>
| |
| - | (35 cycles, annealing in 59℃)<br>
| |
| - | <br>
| |
| - | <h5>digestion → gel extranction</h5><br>
| |
| - | A-7 SP, 4-11L XP, pSB1A2 (B-10), A-7 linear XP<br>
| |
| - | A-9 linear XP, A-10 linear XP, B-1 linear XP<br>
| |
| - | <br>
| |
| - | 1kbp Ladder, A-7 SP, 4-11L XP<br>
| |
| - | (ここに20140814_1を貼付)<br>
| |
| - | (ここに20140814_2を貼付)<br>
| |
| - | OK!<br>
| |
| - | A-10 XP, 100bp Ladder<br>
| |
| - | (ここに20140814_3を貼付)<br>
| |
| - | OK!(low concentration)<br>
| |
| - | <br>
| |
| - | 100bp Ladder, pSB1A2(B-10), pSB1A2(B-10), A-7, A-9, B-1 <br>
| |
| - | (ここに20140814_4を貼付)<br>
| |
| - | (ここに20140814_5を貼付）<br>
| |
| - | Incomplete cut of pSB1A2 is weigh on our mind.<br>
| |
| - | <br>
| |
| - | <h5>gel making</h5><br>
| |
| - | 2% 25mL<br>
| |
| - | 1% 200mL<br>
| |
| - | <br>
| |
| - | <h5>colony PCR</h5><br>
| |
| - | D-7(Ampicillin), D-7(Chloramphenicol)<br>
| |
| - | <br>
| |
| - | 1kbp Ladder, D-7(Amp) #1~4, D-7(CP) #1~4<br>
| |
| - | (ここに20140814_4を貼付)<br>
| |
| - | <br>
| |
| - | <h5>ligation → TF</h5><br>
| |
| - | D-6 (A-7 SP + 4-11L XP), A-7 (linear XP + pSB1A2 XP)<br>
| |
| - | A-9, A-10, B-1 (linear XP + pSB1A2 XP)<br>
| |
| - | <br>
| |
| - | <h3>8,15,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa,Nakashima,Tara,Takemura,Nakamura<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>miniprep</h5><br>
| |
| - | D-7 #3 (made glycerol stock)<br>
| |
| - | <br>
| |
| - | <h5>digestion → gel extraction</h5><br>
| |
| - | A-10 SP, D-7 XP<br>
| |
| - | (ここに20140815_1を貼付)<br>
| |
| - | (ここに20140815_2を貼付)<br>
| |
| - | <br>
| |
| - | <h5>colony PCR</h5><br>
| |
| - | A-7 #1~4, A-9 #1~4, A-10 #1~4, B-1 #1~4, D-6 #1~4<br>
| |
| - | (ここに20140815_3を貼付)<br>
| |
| - | A-7 #3,4, A-10 #1,2,4, B-1 #1,2,4：OK!<br>
| |
| - | <br>
| |
| - | D-6<br>
| |
| - | (ここに20140815_4を貼付)<br>
| |
| - | OK!<br>
| |
| - | <br>
| |
| - | <h5>ligation → TF</h5><br>
| |
| - | E-18 (A-10 SP + D-7 XP)<br>
| |
| - | <br>
| |
| - | <h5>preculture</h5><br>
| |
| - | A-7 #3,4, A-10 #1,2,4, B-1 #1,2,4, D-6 #1<br>
| |
| - | <br>
| |
| - | <h3>8,16,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>miniprep</h5><br>
| |
| - | A-7 #3,4, A-10 #1,2,4, B-1 #1,2,4, D-6 #1<br>
| |
| - | (made glycerol stock)<br>
| |
| - | <br>
| |
| - | 8,18,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa,Hiura<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>digestion → gel extraction</h5><br>
| |
| - | A-1 SP, A-2 SP, A-3 SP, D-6 XP *2, A-7 #3,4, B-10<br>
| |
| - | (ここに20140818_1を貼付)<br>
| |
| - | <br>
| |
| - | <h5>sequence</h5><br>
| |
| - | A-7, A-7 #3,4, A-10, A-10 #1,2,4, B-1 #1,2,4<br>
| |
| - | C-6 F, C-6 R, D-7 F, D-7 R, D-6 F, D-6 R<br>
| |
| - | <br>
| |
| - | <h5>colony PCR</h5><br>
| |
| - | E-18 #1~3<br>
| |
| - | (ここに20140818_2を貼付)<br>
| |
| - | OK!<br>
| |
| - | <br>
| |
| - | <h5>ligation → TF</h5><br>
| |
| - | E-14 (A-3 SP + D-6 XP)<br>
| |
| - | E-15 (A-1 SP + D-6 XP)<br>
| |
| - | E-16 (A-2 SP + D-6 XP)<br>
| |
| - | <br>
| |
| - | <h5>preculture</h5><br>
| |
| - | E-18 #1<br>
| |
| - | <br>
| |
| - | <h3>8,19,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa,Nakamura,Yamanaka,Yoshikawa(Fresh)<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>miniprep</h5><br>
| |
| - | E-18 (made glycerol stock)<br>
| |
| - | <br>
| |
| - | <h5>digestion</h5> <br>
| |
| - | A-8 XP, B-1 #1,2,4 SP<br>
| |
| - | →Today, sequences of these dna proved to be wrong, so we disposed them.<br>
| |
| - | <br>
| |
| - | <h5>making gel</h5><br>
| |
| - | 2% gel:25ml<br>
| |
| - | <br>
| |
| - | <h5>colony PCR</h5><br>
| |
| - | C-5, E-14, E-15,E-16<br>
| |
| - | <br>
| |
| - | <h5>sequence</h5><br>
| |
| - | A-7:RBS<br>
| |
| - | A-7 #3:none<br>
| |
| - | A-7 #4:none<br>
| |
| - | A-10:OK<br>
| |
| - | B-1: all none<br>
| |
| - | <br>
| |
| - | <h3>8,20,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa,Nakamura,Tsukada,Yamanaka,Itoh<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>PCR</h5><br>
| |
| - | sigma11, sigma20, anti11, anti20<br>
| |
| - |
| |
| - | <h5>TF</h5><br>
| |
| - | 1-21P<br>
| |
| - | <br>
| |
| - | <h3>8,21,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa,Nakashima<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>digestion → gel extraction</h5><br>
| |
| - | pSB1C3(A-4), A-6, B-3(We did not extract.), A-6'(NEB buffer)<br>
| |
| - | (ここに20140821_1を貼付)<br>
| |
| - | <br>
| |
| - | <h5>colony PCR</h5><br>
| |
| - | A-2, A-3, A-4, 1-21P, NC<br>
| |
| - | (ここに20140821_2を貼付)<br>
| |
| - | <br>
| |
| - | <h5>ligation → TF</h5><br>
| |
| - | B-3, C-2, C-2'<br>
| |
| - | <br>
| |
| - | <h5>preculture</h5><br>
| |
| - | 1-21P<br>
| |
| - | <br>
| |
| - | <h3>8,22,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa,Nakamura,Nakashima<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>miniprep</h5><br>
| |
| - | 1-21P(made glycerol stock)<br>
| |
| - | <br>
| |
| - | <h5>colony PCR</h5><br>
| |
| - | B-3, C-2, C-2<br>
| |
| - | (ここに10140822_1を貼付)<br>
| |
| - | <br>
| |
| - | <h5>digestion → gel extraction</h5><br>
| |
| - | 1-21P SP, D-7 XP, C-6 E, C-6 P, C-6(NC)<br>
| |
| - | (ここに20140822_2を貼付)<br>
| |
| - | (ここに20140822_3を貼付)<br>
| |
| - | <br>
| |
| - | <h5>ligation → TF</h5><br>
| |
| - | K-1(1-21P SP + D-7 XP)<br>
| |
| - | <br>
| |
| - | <h5>preculture</h5><br>
| |
| - | B-3 #1,4, D-7, C-2 #1,3, C-2' #3,4<br>
| |
| - | <br>
| |
| - | <h3>8,23,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>miniprep</h5><br>
| |
| - | D-7, B-3 #1,4, C-2 #1,3, C-2' #3,4<br>
| |
| - | <br>
| |
| - | <h3>8,25,2014</h3><br>
| |
| - | <h3>Lab member</h3>Nakashima,Nakamura,Yamanaka,Hiura<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>digestion → gel extraction</h5><br>
| |
| - | A-8 EX *2, B-3 #1 ES, B-3 #4 ES, C-2 #1 ES, C-2 #3 ES<br>
| |
| - | (ここに20140825_1を貼付)<br>
| |
| - | (ここに20140825_2を貼付)<br>
| |
| - | <br>
| |
| - | <h5>Plate making</h5><br>
| |
| - | Ampicillin*10, Chloramphenicol*20<br>
| |
| - | <br>
| |
| - | <h5>colony PCR</h5><br>
| |
| - | K-1<br>
| |
| - | (ここに20140825_3を貼付)<br>
| |
| - | #1,4:OK!<br>
| |
| - | <br>
| |
| - | <h5>sequence</h5><br>
| |
| - | B-3 #1,4, C-2 #1,3, C-2' #3,4, E-18, A-2, A-3<br>
| |
| - | <br>
| |
| - | <h5>ligation → TF</h5><br>
| |
| - | D-3 1 (A-8 EX + C-2 #1 ES), D-3 2(A-8 EX + C-2 #3 ES)<br>
| |
| - | C-10 1(A-8 EX + B-3 #1 ES), C-10 2(A-8 EX + B-3 #4 ES)<br>
| |
| - | <br>
| |
| - | <h5>preculture</h5><br>
| |
| - | A-2, A-8, K-1 #1, C-2 #1,3, B-3 #1<br>
| |
| - | <br>
| |
| - | <h3>8,26,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa,Nakashima,Yamanaka,Takemura,Tsukada<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>miniprep</h5><br>
| |
| - | K-1(made glycerol stock), A-2, A-8, B-3 #1, C-2 #1, C-2 #3<br>
| |
| - | <br>
| |
| - | <h5>colony PCR</h5><br>
| |
| - | D-3 (1), D-3 (2), C-10 (1), C-10 (2)<br>
| |
| - | (ここに20140826_1を貼付)<br>
| |
| - | OK!(C-10(1)#4 may be a little shorter.)<br>
| |
| - | <br>
| |
| - | <h5>digestion → gel extraction</h5><br>
| |
| - | K-2 linear EP, K-3 linear EP, K-4 limear EP, K-5 linear EP, pSB1C3 (A-4) EP<br>
| |
| - | (ここに20140826_2を貼付)<br>
| |
| - | We forgot to take the photo before we sliced the band.<br>
| |
| - | <br>
| |
| - | <h5>ligation → TF</h5><br>
| |
| - | K-2, K-3, K-4, K-5<br>
| |
| - |
| |
| - | <h5>preculture</h5><br>
| |
| - | D-3 #1, C-10 #1<br>
| |
| - |
| |
| - | <h5>sequence</h5><br>
| |
| - | A-2:E-X-S-S-Pconst(weak)-S-P wrong <h5>sequence</h5><br>
| |
| - | A-3:OK<br>
| |
| - | B-3 #1:OK<br>
| |
| - | B-3 #4:OK<br>
| |
| - | C-2 #1:OK<br>
| |
| - | C-2 #3:OK<br>
| |
| - | C-2' #3:point mutation *2<br>
| |
| - | C-2' #4:OK<br>
| |
| - | E-18:OK<br>
| |
| - | <br>
| |
| - | <h3>8,27,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa,Nakashima,Yoshikawa(fresh),Itoh,Tara,Nakamura<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>miniprep</h5><br>
| |
| - | D-3, C-10(made glycerol stock)<br>
| |
| - | <h5>digestion → gel extraction</h5><br>
| |
| - | A-1 SP, A-3 SP, A-10 SP, D-3 XP *2<br>
| |
| - | (ここに20140827_1を貼付)<br>
| |
| - | (ここに20140827_2を貼付)<br>
| |
| - | D-3 is a little strange.Contamination?<br>
| |
| - | <br>
| |
| - | <h5>colony PCR</h5><br>
| |
| - | K-2~5 #1~4<br>
| |
| - | *100bp Ladder<br>
| |
| - | (ここに20140827_3を貼付)<br>
| |
| - | 500bp band:self ligation of backbone(A-4 200bop) <br>
| |
| - | 300bp band:If this band is blank vector, this band(EP cut) must be shorter than 300bp.<br>
| |
| - | So, this band is OK!.<br>
| |
| - | →K-2 #4, K-3 #1, K-4 #1,2, K-5 #2,3,4:OK!<br>
| |
| - | <br>
| |
| - | <h5>ligation → TF</h5><br>
| |
| - | E-5(A-3 SP + D-3 XP), E-6(A-1 SP + D-3 XP), E-20(A-10 SP + D-3 XP)<br>
| |
| - | <br>
| |
| - | <h5>preculture</h5><br>
| |
| - | E-15<br>
| |
| - | D-3, C-10(Recovery)<br>
| |
| - | K-2 #4, K-3 #1, K-5 #2<br>
| |
| - | <br>
| |
| - | <h5>Remarks</h5><br>
| |
| - | (ここに20140827_4を貼付)<br>
| |
| - | We observed that <i>pCMV</i> expressed in <i>Escherichia.coli</i>.<br>
| |
| - | Left:<i>pCMV-GFP</i><br>
| |
| - | Right:<i>pConst(strong)-GFP</i><br>
| |
| - | We may be able to carry out the characterization of <i>pCMV</i> and meet the gold medal requirement(parts implovement).<br>
| |
| - | <br>
| |
| - | <h3>8,28,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa,Nakashima,Itoh,Tara,Tsukada,Yamanaka<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>miniprep</h5><br>
| |
| - | K-2~5, E-23(made glycerol stock)<br>
| |
| - | C-10, D-3<br>
| |
| - | <br>
| |
| - | <h5>digestion →gel extraction</h5><br>
| |
| - | K-2 EX, K-3 EX, K-4 EX, K-5 EX, E-23 EP, C-6 ES *2, pSB1C3(A-4) EP, pSB1C3(A-4) XP<br>
| |
| - | (ここに20140828_1を貼付)<br>
| |
| - | (ここに20140828_2を貼付)<br>
| |
| - | →pSB1C3 XP:keep in freezer<br>
| |
| - | <br>
| |
| - | <h5>overlap extension PCR</h5><br>
| |
| - | A-7, A-9, B-1, B-2, D-8, D-9<br>
| |
| - | <br>
| |
| - | <h5>check & colony PCR</h5><br>
| |
| - | check:E-5, E-6<br>
| |
| - | colony PCR:(E-5 #1~4, E-6 #1~4, NC), A-7, A-9, D-8, B-1, B-2, D-9<br>
| |
| - | (ここに20140828_3を貼付)<br>
| |
| - | A-7, A-9, B-1, D-8:OK!<br>
| |
| - | B-2, D-9:Needs retry in higher concentration.<br>
| |
| - | E-5 #2~4, E-6:OK!<br>
| |
| - | <h5>ligation → TF</h5><br>
| |
| - | E-23'(E-23 EP + pSB1C3 EP), L-1~4(C-6 ES + K-2~5 EX)<br>
| |
| - | <br>
| |
| - | <h3>8,29,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa,Nakashima,Nakamura,Tara,Tsukada,Yamanaka<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>miniprep</h5><br>
| |
| - | E-5(made glycerol stock)<br>
| |
| - | A-4, C-6<br>
| |
| - | E-6:No colony<br>
| |
| - | <br>
| |
| - | <h5>digestion →gel extraction</h5><br>
| |
| - | A-7 linear XP, A-9 linear XP, A-10 SP, B-1 linear XP, D-3 XP, D-8 linear XP, E-5 SP, E-18 XP<br>
| |
| - | (ここに20140829_1を貼付)<br>
| |
| - | (ここに20140829_2を貼付)<br>
| |
| - | <br>
| |
| - | <h5>PCR</h5><br>
| |
| - | B-2, D-9(retry)<br>
| |
| - | <br>
| |
| - | <h5>check & colony PCR</h5><br>
| |
| - | E-23' #1~4, B-2, D-9<br>
| |
| - | (ここに20140829_3を貼付)<br>
| |
| - | L-1~4<br>
| |
| - | (ここに20140829_4を貼付)<br>
| |
| - | E-23 #1, L-1 #2,4, L-2 #4, L-3 #1~3, L-4 #2,4:OK!<br>
| |
| - | PCR:failed<br>
| |
| - | <br>
| |
| - | <h5>sequence</h5> order<br>
| |
| - | E-23, K-1, K-2~5<br>
| |
| - | <br>
| |
| - | <h5>ligation → TF</h5><br>
| |
| - | A-7, A-9, B-1, D-8( linear XP + pSB1C3 XP)<br>
| |
| - | F-2 (E-18 XP + E-5 SP), E-20(A-10 SP + D-3 XP) <br>
| |
| - | <br>
| |
| - | <h5>preculture</h5><br>
| |
| - | A-10, E-6, E-18, E-23', K-3, L-1~4<br>
| |
| - | <br>
| |
| - | <h3>8,30,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>miniprep</h5><br>
| |
| - | A-10, E-18, K-3<br>
| |
| - | L-1~4, E-23', E-6, E-18(made glycerol stock)<br>
| |
| - | <br>
| |
| - | <h5>PCR</h5><br>
| |
| - | B-2, D-9<br>
| |
| - | <br>
| |
| - | <h3>9,1,2014</h3><br>
| |
| - | <h3>Lab member</h3>Nakashima,Itoh,Tara,Tsukada,Takemura<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>PCR clean up</h5><br>
| |
| - | B-2, D-9<br>
| |
| - | (ここに20140901_1を貼付)<br>
| |
| - | failed<br>
| |
| - | <br>
| |
| - | <h5>digestion → gel extraction</h5><br>
| |
| - | L-4 ES, E-6 ES, E-18 EX, L-1~3 ES, K-2~5 EX<br>
| |
| - | <br>
| |
| - | (ここに20140901_2を貼付)<br>
| |
| - | (ここに20140901_3を貼付)<br>
| |
| - | <h5>Making plate</h5><br>
| |
| - | CP *30<br>
| |
| - | <br>
| |
| - | <h5>colony PCR</h5><br>
| |
| - | A-7 #1~4, A-9 #1<br>
| |
| - | (ここに20140901_4を貼付)<br>
| |
| - | OK!<br>
| |
| - | D-8 #1~4, E-20 #1~4, F-2 #1~4 (100bp Ladder)<br>
| |
| - | (ここに20140901_5を貼付)<br>
| |
| - | D-8 #1~4, E-20 #1~4,F-2#1,4:OK!<br>
| |
| - | B-1 #1~4<br>
| |
| - | (ここに20140901_6を貼付)<br>
| |
| - | OK!<br>
| |
| - | <br>
| |
| - | <h5>Ligation</h5> <br>
| |
| - | G-5 (E-6 ES + E-18 EX), M-1~4(K-2~5 EX + L-1~4 ES)<br>
| |
| - | <br>
| |
| - | <h5>preculture</h5><br>
| |
| - | A-7 #1~4, A-9 #1, B-1 #1~4, D-8 #1~4, F-2, E-20<br>
| |
| - | <br>
| |
| - | <h3>9,2,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa,Nakashima,Hiura,Tara,Tsukada,Takemura<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>miniprep</h5><br>
| |
| - | A-7 #1~4, A-9, B-1 #1~4, D-8 #1~4, F-2, E-2(made glycerol stock)<br>
| |
| - | <br>
| |
| - | <h5>digestion → gel extraction</h5><br>
| |
| - | A-4 SP, A-7 SP #1, A-7 SP #2, A-9 SP<br>
| |
| - | (ここに20140902_1を貼付)<br>
| |
| - | (ここに20140902_2を貼付)<br>
| |
| - | A-8XP<br>
| |
| - | (ここに20140902_3を貼付)<br>
| |
| - | (ここに20140902_4を貼付)<br>
| |
| - | <br>
| |
| - | B-1 #1, B-1 #2, B-3 XP, B-10 XP, D-7 XP, D-8 XP, E-20 XP, F-2 SP<br>
| |
| - | (ここに20140902_5を貼付)<br>
| |
| - | (ここに20140902_6を貼付)<br>
| |
| - | D-8:???<br>
| |
| - | <br>
| |
| - | <h5>sequence</h5><br>
| |
| - | A-7 #1~4, B-1 #1~4<br>
| |
| - | D-8 #1~4<br>
| |
| - | A-9, A-4, E-5, E-6, E-20<br>
| |
| - | <br>
| |
| - | <h5>PCR</h5><br>
| |
| - | B-2, D-9<br>
| |
| - | <br>
| |
| - | <h5>check & colony PCR</h5><br>
| |
| - | M-1~3 #1~4<br>
| |
| - | (ここに20140902_7を貼付)<br>
| |
| - | OK!<br>
| |
| - | M-4 #1~4, B-2, D-9, PC(VF2→B-10←VR )<br>
| |
| - | NC, G-5<br>
| |
| - | (ここに20140902_8を貼付)<br>
| |
| - | M-4 #1~4,PC(VF2→B-10←VR ):OK!<br>
| |
| - | (M-4 #2~4 is a little longer?)<br>
| |
| - | <br>
| |
| - | <h5>ligation → TF</h5><br>
| |
| - | C-4(A-7 SP + B-3 XP), C-5(A-7 SP + B-10 XP), E-17(A-9 SP + D-7 XP)<br>
| |
| - | D-1(B-1 SP + D-8 XP), E-21(A-4 SP + D-8 XP), J-4 (F-2 SP + E-20 XP)<br>
| |
| - | <br>
| |
| - | <h5>preculture</h5><br>
| |
| - | M-1~4 #1, G-5 #1, E-20<br>
| |
| - | <br>
| |
| - | <h5>We got a result!!</h5><br>
| |
| - | simpler version of assay 1.<br>
| |
| - | Left:Pσ11→GFP generator<br>
| |
| - | Right:Pconst(strong)→σ11 generator & Pσ11→GFP generator<br>
| |
| - | (ここに20140902_9を貼付)<br>
| |
| - | <br>
| |
| - | <h3>9,3,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa,Nakashima,Hiura,Tara,Yamanaka,Takemura<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | M-1~4, G-5(made glycerol stock of M-3,4)<br>
| |
| - | <br>
| |
| - | <h5>sequence</h5><br>
| |
| - | A-4: OK<br>
| |
| - | A-7: all OK<br>
| |
| - | A-9: OK<br>
| |
| - | B-1:all OK<br>
| |
| - | D-8#1: NG (2 skip)<br>
| |
| - | #2; OK<br>
| |
| - | #3: NG (1 mut.)<br>
| |
| - | #4; NG (2 mut. 2 skip)<br>
| |
| - | E-5: OK<br>
| |
| - | E-6: OK<br>
| |
| - | E-20:OK<br>
| |
| - | <br>
| |
| - | <h5>digestion → gel extraction</h5><br>
| |
| - | A-7 #1, 2 SP, C-9 XP, C-10 XP, K-2~4 EX, M-1~4 ES<br>
| |
| - | (ここに20140903_1を貼付)<br>
| |
| - | (ここに20140903_2を貼付)<br>
| |
| - | <br>
| |
| - | <h5>colony PCR</h5><br>
| |
| - | C-4 a, C-5 a, D-1 a, E-17<br>
| |
| - | (ここに20140903_3を貼付)<br>
| |
| - | C-4 a#2,3, C-5 a, D-1 a, E-17:OK!<br>
| |
| - | E-21 b, J-4<br>
| |
| - | (ここに20140903_4を貼付)<br>
| |
| - | J-4:wrong<br>
| |
| - | E-21:a little shorter<br>
| |
| - | <br>
| |
| - | <h5>PCR</h5><br>
| |
| - | B-2, D-9, B-7, B-2(Taq), D-9(Taq)
| |
| - | Taq:positive control<br>
| |
| - | anealing temperature:51℃～61℃(gradient)<br>
| |
| - | <br>
| |
| - | <h5>ligation → TF</h5><br>
| |
| - | D-5 (A-7 SP + C-10 XP), D-6 (A-7 SP + C-9 XP), N-1~4 (M-1~4 ES + K-2~5 EX)<br>
| |
| - | N-5 (M-1 ES K-3 EX), N-6 (M-3 ES + K-5 EX)<br>
| |
| - | <br>
| |
| - | <h3>9,4,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa,Nakashima,Nakamura,Tara,Yamanaka,Tsukada<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>miniprep</h5><br>
| |
| - | E-20, D-1(made glycerol stock)<br>
| |
| - | <br>
| |
| - | <h5>digestion → gel extraction</h5><br>
| |
| - | pSB1C3 (A-4) XS, pSB1C3 (A-4) XP, A-3 SP, D-1 XP<br>
| |
| - | (ここに20140904_1を貼付)<br>
| |
| - | (ここに20140904_2を貼付)<br>
| |
| - | <br>
| |
| - | <h5>PCR check & clean up</h5><br>
| |
| - | D-9 #1,3,5,7,9
| |
| - | (ここに20140904_3を貼付)<br>
| |
| - | There are bands in #5,7,9<br>
| |
| - | B-2 1,3,5,7,9,11, B-7.<br>
| |
| - | (ここに20140904_4を貼付)<br>
| |
| - | There are bands in B-2 #5,7, B-7.<br>
| |
| - | B-2 4,6, D-9 10,11,12<br>
| |
| - | (ここに20140904_5を貼付)<br>
| |
| - | OK!<br>
| |
| - | We decided to clean up B-2 #5, D-9 #11, B-7.
| |
| - | <br>
| |
| - | <h5>Making Plate</h5><br>
| |
| - | CP *30<br>
| |
| - | <br>
| |
| - | <h5>digestion</h5><br>
| |
| - | B-2 linear SP, D-9 linear XP, B-7 linear XP<br>
| |
| - | <br>
| |
| - | <h5>colony PCR</h5><br>
| |
| - | D-5, D-6, N-1, N-2 #1~4<br>
| |
| - | (ここに20140904_6を貼付)<br>
| |
| - | D-5, D-6, N-1, N-2 #1,2:OK!<br>
| |
| - | N-3~6 #1~4<br>
| |
| - | (ここに20140904_7を貼付)<br>
| |
| - | N-3,N-4#1,3,4,N-5#1,4,N-6:OK!<br>
| |
| - | <br>
| |
| - | <h5>ligation → TF</h5><br>
| |
| - | E-1 (A-3 SP + D-1 XP), B-2 (B-2 linear XP + pSB1C3 XP), D-9 (D-9 linear XP + pSB1C3 XP)<br>
| |
| - | B-7 (B-7 linear XP + pSB1C3 XP), Emp. (pSB1C3 XS)<br>
| |
| - | <br>
| |
| - | <h5>miniprep</h5><br>
| |
| - | M-1, M-2, C-4, C-5, E-17, E-21, J-4 (made glycerol stock)<br>
| |
| - | K-2, K-5, C-9<br>
| |
| - | <br>
| |
| - | <h5>preculture</h5><br>
| |
| - | F-2, D-5, D-6, N-1~6<br>
| |
| - | <br>
| |
| - | <h3>9,5,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa,Nakashima,Nakamura,Takemura,Yamanaka<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>miniprep</h5><br>
| |
| - | F-2<br>
| |
| - | N-1~6, D-5, D-6<br>
| |
| - | →N-1, 2, 4, 5. 6 :failed<br>
| |
| - | <br>
| |
| - | <h5>digestion → gel extraction</h5><br>
| |
| - | A-1 SP, A-3 SP, D-5 XP, D-6 SP, E-20 XP, K-4 EX, N-3 ES, F-2 SP<br>
| |
| - | J-4 E(check)<br>
| |
| - | (ここに20140905_1を貼付)<br>
| |
| - | (ここに20140905_2を貼付)<br>
| |
| - | <br>
| |
| - | <h5>ligation → TF</h5><br>
| |
| - | E-11 (A-3 SP + D-5 XP), E-12 (A-1 SP + D-5 XP), E-14 (A-3 SP + D-6 SP)<br>
| |
| - | E-15 (A-1 SP + D-6 XP), O-3 (K-4 EX + N-3 ES)<br>
| |
| - | <br>
| |
| - | <h5>PCR</h5><br>
| |
| - | B-2, D-9, B-7<br>
| |
| - | <br>
| |
| - | <h5>colony PCR</h5><br>
| |
| - | B-2, B-7, D-9, E-1 #1~4<br>
| |
| - | (ここに20140905_3を貼付)<br>
| |
| - | B-7 #2,3, D-9 #1, E-1 #3,4:OK!<br>
| |
| - | Z-1 #2~4<br>
| |
| - | (ここに20140905_3を貼付)<br>
| |
| - | <br>
| |
| - | <h5>preculture</h5><br>
| |
| - | B-7 #2,3, D-9 #1, E-1 #3, Z-1 #2, K-3, N-1,2,4~6<br>
| |
| - | <br>
| |
| - | <h3>9,6,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa,Nakashima<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>miniprep</h5><br>
| |
| - | B-7 #2,3, D-9 #1, E-1 #3, Z-1 #2 (made glycerol stock)<br>
| |
| - | N-1,2,4~6, K-3<br>
| |
| - | <br>
| |
| - | <h5>PCR</h5><br>
| |
| - | B-2, D-9, B-7<br>
| |
| - | <br>
| |
| - | <h5>check & colony PCR</h5><br>
| |
| - | B-2, D-9, B-7, B-2#5~8, B-7 #2,3,5,6, D-9 #5,6.7,8<br>
| |
| - | (ここに20140906_1を貼付)<br>
| |
| - | <br>
| |
| - | <h3>9,8,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa,Nakashima,Nakamura,Takemura,Yamanaka,Tara<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>digestion → gel extaction</h5><br>
| |
| - | B-2 linear XP (did not extracted)<br>
| |
| - | N-1,2,4~6 ES<br>
| |
| - | (ここに20140908_1を貼付)<br>
| |
| - | (ここに20140908_2を貼付)<br>
| |
| - | A-4 SP, pSB1C3 (A-4) XP, A-6 SP, B-7 #2 XP, B-7 #3 XP, D-9 #1 XP, K-2,3,5 EX<br>
| |
| - | (ここに20140908_3を貼付)<br>
| |
| - | B-7 #2, D-9 #1:disposed(incomplete cut)<br>
| |
| - | B-7 #3:disposed(a little longer)(This band proved to be correct.9/9 wrote)<br>
| |
| - | (ここに20140908_4を貼付)<br>
| |
| - | <br>
| |
| - | <h5>colony PCR</h5><br>
| |
| - | E-11,12,14,15 #1~4<br>
| |
| - | (ここに20140908_5を貼付)<br>
| |
| - | OK!<br>
| |
| - | O-3 #1,2<br>
| |
| - | (ここに20140908_6を貼付)<br>
| |
| - | #2:OK!<br>
| |
| - | <br>
| |
| - | <h5>PCR</h5><br>
| |
| - | B-2<br>
| |
| - | (ここに20140908_7を貼付)<br>
| |
| - | B-7,D-9<br>
| |
| - | (ここに20140908_8を貼付)<br>
| |
| - | The band of the longest DNA is OK!<br>
| |
| - | <br>
| |
| - | <h5>ligation → TF</h5><br>
| |
| - | O-1 (N-1 ES + K-2 EX), O-2 (N-2 ES + K-3 EX), O-4 (N-4 ES + K-5 EX), O-5 (N-5 ES + K-3 EX)<br>
| |
| - | O-6 (N-6 ES + K-5 EX), B-2 (B-2 linear XP (9/8 <h5>PCR</h5>) + pSB1C3 XP)<br>
| |
| - | B-2' (B-2 linear XP + pSB1C3 XP), D-9, B-7 <br>
| |
| - | <br>
| |
| - | <h5>preculture</h5><br>
| |
| - | D-5, D-6, N-3, E-21, A-3, K-4<br>
| |
| - | E-11 #1, E-12 #1, E-14 #1, E-15 #1, O-3 #2, B-7 #6, D-9 #5,6<br>
| |
| - | <br>
| |
| - | <h3>9,9,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa,Nakashima,Nakamura,Takemura,Yamanaka<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>miniprep</h5><br>
| |
| - | E-11,12,14,15, O-3, B-7 #6, D-9 #5,6 (made glycerol stock)<br>
| |
| - | D-5, N-3, K-4, A-3<br>
| |
| - | D-6:did not grow<br>
| |
| - | D-5:diposed(doubt of cross contamination)<br>
| |
| - | <br>
| |
| - | <h5>digestion → gel extraction</h5><br>
| |
| - | B-2 linear XP (did not extracted)<br>
| |
| - | pSB1C3 (A-4) XP, A-6 SP, A-8 EX, B-7 #6 XP, D-9 linear XP, B-7 linear , E-1 EX, E-14 ES, E-15 ES, O-3 ES<br>
| |
| - | (ここに20140909_1を貼付)<br>
| |
| - | (ここに20140909_2を貼付)<br>
| |
| - | <br>
| |
| - | <h5>colony PCR</h5><br>
| |
| - | B-2 #1~3, B-2' #1~4, B-7 #1~3, D-9 #1,2, O-1 #1~4<br>
| |
| - | (ここに20140909_3を貼付)<br>
| |
| - | B-2' #1, B-7 #1,2, O-1:OK!<br>
| |
| - | O-2,4,5,6 #1~4<br>
| |
| - | (ここに20140909_4を貼付)<br>
| |
| - | O-2,3,4,6,O-5#2,3,4<br>
| |
| - | <br>
| |
| - | <h5>sequence</h5><br>
| |
| - | B-7 #2: NG<br>
| |
| - | B-7 #3: OK<br>
| |
| - | D-9 #1: NG<br>
| |
| - | E-1: OK<br>
| |
| - | C-4: OK<br>
| |
| - | C-5: OK<br>
| |
| - | D-5: OK<br>
| |
| - | D-6: OK<br>
| |
| - | E-17: OK<br>
| |
| - | E-21: OK<br>
| |
| - | F-2: ?<br>
| |
| - | J-4: OK<br>
| |
| - | N-1: OK<br>
| |
| - | N-2: M-2<br>
| |
| - | N-3: OK<br>
| |
| - | N-4: M-4<br>
| |
| - | N-5: OK<br>
| |
| - | N-6: OK<br>
| |
| - | <br>
| |
| - | <h5>ligation → TF</h5><br>
| |
| - | D-9 (D-9 linear XP + pSB1C3 XP) *2, B-2 (B-2 linear XP + pSB1C3 XP)*2<br>
| |
| - | P-3 (O-3 ES + A-8 EX), I-1 (E-14 ES + E-1 EX), I-2 (E-15 ES + E-1 EX)<br>
| |
| - | <br>
| |
| - | <h5>preculture</h5><br>
| |
| - | F-2, D-5,6, D-9 #5,6, E-12, K-4<br>
| |
| - | O-1,2,4,6 #1, O-5 #2, B-2' #1<br>
| |
| - | <br>
| |
| - | <h3>9,10,2014</h3><br>
| |
| - | <h3>Lab member</h3>Nakashima,Hiura,Takemura,Yamanaka,Yoshikawa(Fresh)<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>miniprep</h5><br>
| |
| - | O-1,5,6, N-2,4, F-2, B-2' #1 (made glycerol stock)<br>
| |
| - | E-12, K-4, D-5<br>
| |
| - | D-6:did not grow<br>
| |
| - | <br>
| |
| - | <h5>digestion → gel extraction</h5><br>
| |
| - | A-6 SP, A-7 SP, A-8 EX, B-2' XP, B-7 XP<br>
| |
| - | (ここに20140910_1を貼付)<br>
| |
| - | (ここに20140910_2を貼付)<br>
| |
| - | K-3 EX, K-5 EX, O-1 ES, N-2 ES, N-4 ES<br>
| |
| - | (ここに20140910_3を貼付)<br>
| |
| - | (ここに20140910_4を貼付)<br>
| |
| - | O-5 ES, O-6 ES<br>
| |
| - | (ここに20140910_5を貼付)<br>
| |
| - | (ここに20140910_6を貼付)<br>
| |
| - | <br>
| |
| - | <h5>colony PCR</h5><br>
| |
| - | I-1 #1~4<br>
| |
| - | (ここに20140910_7を貼付)<br>
| |
| - | B-2(140909) #1~8, D-9 #1~8<br>
| |
| - | (ここに20140910_8を貼付)<br>
| |
| - | B-2 #2, D-9 all, I-1 #2~4, I-2 #1~4:OK!<br>
| |
| - | <br>
| |
| - | <h5>ligation → TF</h5><br>
| |
| - | O-2 (N-2 ES + K-3 EX), O-4 (N-4 ES + K-5 EX), P-1 (O-1 ES + A-8 EX), P-5 (O-5 ES + A-8 EX)<br>
| |
| - | P-6 (O-6 ES + A-8 EX), C-1' (B-2' XP + A-6 SP), C-3' (B-2 ' XP + A-7 SP), C-8 (B-7 XP + A-6 SP)<br>
| |
| - | <br>
| |
| - | <h5>preculture</h5><br>
| |
| - | D-9 #5,6, D-9(140909) #1~4, B-2 (140909) #2, I-1 #2, I-2 #1, D-6 #1<br>
| |
| - | <br>
| |
| - | <h3>9,11,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa,Nakashima,Nakamura,Hiura,Itoh,Tsukada<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>miniprep</h5><br>
| |
| - | D-9 #1~4, B-2 #2, I-1, I-2, D-6 (made glycerol stock)<br>
| |
| - | <br>
| |
| - | <h5>digestion → gel extraction</h5><br>
| |
| - | A-1 EP, pSB1C3(A-3) EP *2, A-4 SP, A-6 SP, A-7 SP, A-8 EX, A-10 EX, B-2 #2 XP, E-6 EP, E-12 EP<br>
| |
| - | (ここに20140911_1を貼付)<br>
| |
| - | (ここに20140911_2を貼付)<br>
| |
| - | E-15 EP, E-18 EP, E-20 EP, D-9 #1 XP, O-3 ES<br>
| |
| - | (ここに20140911_3を貼付)<br>
| |
| - | (ここに20140911_4を貼付)<br>
| |
| - | <br>
| |
| - | <h5>sequence</h5><br>
| |
| - | B-2' #1, B-2 #2, F-2, E-11, E-12, E-14, E-15, O-1, N-2, O-3, N-4, O-5, O-6, D-9 #1~4<br>
| |
| - | <br>
| |
| - | <h5>Plate Making</h5><br>
| |
| - | CP *30<br>
| |
| - | <br>
| |
| - | <h5>colony PCR</h5><br>
| |
| - | C-1', C-3', C-8, O-2, O-4 #1~3<br>
| |
| - | (ここに20140911_5を貼付)<br>
| |
| - | C-1', C-3', C-8, O-2, O-4 #1,2:OK!<br>
| |
| - | P-1, P-5, P-6 #1~3<br>
| |
| - | (ここに20140911_6を貼付)<br>
| |
| - | →P-1 #1~3, P-5 #2, P-6 #1~3:OK!<br>
| |
| - | <br>
| |
| - | <h5>ligation → TF</h5><br>
| |
| - | C-1 (A-6 SP + B-2 #2 XP), C-3 (A-7 SP + B-2 #2 XP)<br>
| |
| - | E-22a (A-4 SP + D-9 #1 XP), E-22b (A-4 SP + D-9 #2 XP), P-3 (O-3 ES + A-8 EX)<br>
| |
| - | A-10 CP, A-1 CP, E-6 CP, E-12 CP, E-20 CP, E-15 CP (replace backbone to pSB1C3)<br>
| |
| - | <br>
| |
| - | <h5>preculture</h5><br>
| |
| - | C-1' #1, C-3' #1, C-8 #1, O-2 #1, O-4 #1, P-1 #1, P-5 #2, P-6 #1, E-12,14,15,18, A-1, O-1,3,5<br>
| |
| - | <br>
| |
| - | <h3>9,12,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa,Nakashima,Hiura,Itoh,Tsukada,Tara<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>miniprep</h5><br>
| |
| - | C-1', C-3', O-2, O-4, P-1, P-5, P-6 (made glycerol stock)<br>
| |
| - | E-12, E-14, E-15, O-1, O-3, O-5, E-18, A-1<br>
| |
| - | <br>
| |
| - | <h5>digestion → gel extraction</h5><br>
| |
| - | A-6 SP, A-8 EX *2, B-2 XP, C-1' ES, C-3' ES, C-8 ES, K-6 SP, O-2 ES, O-3 ES, O-4 ES<br>
| |
| - | (ここに20140912_1を貼付)<br>
| |
| - | (ここに20140912_2を貼付)<br>
| |
| - | C-1,3:did not extracted<br>
| |
| - | P-1 XP, P-5 XP, P-6 XP<br>
| |
| - | (ここに20140912_3を貼付)<br>
| |
| - | (ここに20140912_4を貼付)<br>
| |
| - | <br>
| |
| - | <h5>colony PCR</h5><br>
| |
| - | A-1 CP, A-10 CP, C-3, E-6 CP #1~3<br>
| |
| - | (ここに20140912_5を貼付)<br>
| |
| - | E-12 CP, E-15 CP, E-20 CP, E-22a, E-22b<br>
| |
| - | (ここに20140912_6を貼付)<br>
| |
| - | except E-20#1,2:OK!<br>
| |
| - | <br>
| |
| - | <h5>sequence</h5><br>
| |
| - | B-2' #1: NG<br>
| |
| - | B-2 #2: OK<br>
| |
| - | D-9 #1: NG<br>
| |
| - | #2: NG<br>
| |
| - | #3: NG<br>
| |
| - | #4: OK<br>
| |
| - | O-1: OK<br>
| |
| - | O-3: OK<br>
| |
| - | O-5: OK<br>
| |
| - | O-6: OK<br>
| |
| - | N-2: OK<br>
| |
| - | N-4: OK<br>
| |
| - | E-11: Ok<br>
| |
| - | E-12: OK<br>
| |
| - | E-14: OK<br>
| |
| - | E-15: OK<br>
| |
| - | F-2: OK<br>
| |
| - | <br>
| |
| - | <h5>ligation → TF</h5><br>
| |
| - | C-1 (A-6 SP + B-2 XP), P-2~4 (O-2~4 ES + A-8 EX), Q-1,5,6 (P-1,5,6 XP + K-6 SP)<br>
| |
| - | <br>
| |
| - | <h5>ligation</h5><br>
| |
| - | P-3 (O-3 ES + A-8 EX)<br>
| |
| - | <br>
| |
| - | <h5>preculture</h5><br>
| |
| - | A-4, A-6, A-8, C-3 #1, A-1 CP #1, E-12 CP #1, E-15 CP #1, E-20 CP #3, E-6 CP #1 <br>
| |
| - | <br>
| |
| - | </h3>9,13,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>miniprep</h5><br>
| |
| - | A-4, A-6, A-8
| |
| - | C-3, E-6 CP, E-12 CP, E-15 CP, E-20 CP, A-1 CP(made glycerol stock)<br>
| |
| - | <br>
| |
| - | <h3>9,15,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa,Nakashima,Takemura,Nakamura,Yamanaka,Tara<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>digestion → gel extraction</h5><br>
| |
| - | A-8 EX, C-3 ES<br>
| |
| - | (ここに20140915_1を貼付)<br>
| |
| - | (ここに20140915_2を貼付)<br>
| |
| - | <br>
| |
| - | <h5>colony PCR</h5><br>
| |
| - | C-1 (σ20F, VR)<br>
| |
| - | (ここに20140915_3を貼付)<br>
| |
| - | OK!<br>
| |
| - | <h5>ligation → TF</h5><br>
| |
| - | D-4 (C-3 ES + A-8 EX) *2 (new and old competent cell)<br>
| |
| - | <br>
| |
| - | <h5>preculture</h5><br>
| |
| - | C-1 #1<br>
| |
| - | <br>
| |
| - | <h3>9,16,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa,Nakashima,Hiura,Itoh,Nakamura,Yamanaka<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>miniprep</h5><br>
| |
| - | C-1 (made glycerol stock)<br>
| |
| - | <br>
| |
| - | <h5>colony PCR</h5><br>
| |
| - | D-4, P-2~4<br>
| |
| - | (ここに20140916_1を貼付)<br>
| |
| - | A-10<br>
| |
| - | (ここに20140916_2を貼付)<br>
| |
| - | <br>
| |
| - | Q-1,5,6:confirmed by fluorescence
| |
| - | <br>
| |
| - | <h5>digestion → gel extraction</h5><br>
| |
| - | A-4 SP, A-8 EX, C-1 ES, D-9 XP, E-18 EP, pSB1C3 (A-4) EP<br>
| |
| - | (ここに20140916_3を貼付)<br>
| |
| - | <br>
| |
| - | <h5>ligation → TF</h5><br>
| |
| - | D-2 (A-8 EX + C-1 ES), E-18 CP (E-18 EP + pSB1C3 EP) *2, E-22 (A-4 SP + D-9 XP)<br>
| |
| - | <br>
| |
| - | <h5>preculture</h5><br>
| |
| - | D-4, P-2~4, A-10 CP, Q-1,5,6<br>
| |
| - | <br>
| |
| - | <h5>assay</h5><br>
| |
| - | (ここに20140916_4を貼付)<br>
| |
| - | PC (E-23': <i>Pconst (strong)-GFP-d.term</i>)<br>
| |
| - | Experiment (K-1: <i>pCMV-GFP-d.term</i>)<br>
| |
| - | NC (Z-1: pSB1C3)<br>
| |
| - | absorbance:600nm and 395nm
| |
| - | Absorbance of 395nm proved not to be able to measure.<br>
| |
| - | We need fluorospectro-photometer.<br>
| |
| - | <br>
| |
| - | <h3>9,17,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa,Nakashima,Hiura,Takemura,Yoshikawa(Fresh),Yamanaka<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>miniprep</h5><br>
| |
| - | D-4, P-2~4, Q-1,5,6, A-10 CP (made glycerol stock)<br>
| |
| - | <br>
| |
| - | <h5>digestion → gel extraction</h5><br>
| |
| - | A-1 CP SP, A-3 SP, D-4 XP, K-6 SP, P-2 XP, P-3 XP, P-4 XP<br>
| |
| - | (ここに20140917_1を貼付)<br>
| |
| - | (ここに20140917_2を貼付)<br>
| |
| - | <br>
| |
| - | <h5>colony PCR</h5><br>
| |
| - | D-2, E-18 CP, E-22<br>
| |
| - | (ここに20140917_3を貼付)<br>
| |
| - | <br>
| |
| - | <h5>ligation → TF</h5><br>
| |
| - | E-8 *2 (A-3 SP + D-4 XP), E-9 (A-1 CP SP + D-4 XP), Q-2~4 (K-6 SP + P-2~4 XP)<br>
| |
| - | <br>
| |
| - | <h5>assay</h5><br>
| |
| - | Photo of plate(n=4)<br>
| |
| - | (ここに20140917_4を貼付)<br>
| |
| - | (ここに20140917_5を貼付)<br>
| |
| - | (ここに20140917_6を貼付)<br>
| |
| - | (ここに20140917_7を貼付)<br>
| |
| - | (ここに20140917_8を貼付)<br>
| |
| - | (ここに20140917_9を貼付)<br>
| |
| - | (ここに20140917_10を貼付)<br>
| |
| - | (ここに20140917_11を貼付)<br>
| |
| - | <br>
| |
| - | <h3>9,18,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa,Nakashima,Hiura,Tsukada,Nakamura<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>miniprep</h5><br>
| |
| - | D-2, E-18 CP, E-22<br>
| |
| - | (made glycerol stock)<br>
| |
| - | <br>
| |
| - | <h5>digestion → gel extraction</h5><br>
| |
| - | A-1 CP SP, A-3 SP, A-9 SP, D-2 XP, E-22 SP, G-5 XP<br>
| |
| - | (ここに20140918_1を貼付)<br>
| |
| - | (ここに20140918_2を貼付)<br>
| |
| - | <br>
| |
| - | <h5>colony PCR</h5><br>
| |
| - | E-8, E-9 #1~4<br>
| |
| - | (ここに20140918_3を貼付)<br>
| |
| - | Q-2~4 :confirmed by fluorescent<br>
| |
| - | <br>
| |
| - | <h5>ligation → TF</h5><br>
| |
| - | E-2 (A-3 SP + D-2 XP)<br>
| |
| - | E-3 (A-1CP SP + D-2 XP)<br>
| |
| - | E-19 (A-9 SP + D-2 XP)<br>
| |
| - | H-5 (E-22 SP + G-5 XP)<br>
| |
| - | <br>
| |
| - | <h5>preculture</h5><br>
| |
| - | E-8,9, Q-2~4<br>
| |
| - | <br>
| |
| - | <h3>9,19,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa,Nakashima,Hiura,Tara,Yamanaka,Nakamura<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>miniprep</h5><br>
| |
| - | E-8,9, Q-2~4<br>
| |
| - | <br>
| |
| - | <h5>digestion → gel extraction</h5><br>
| |
| - | A-9 SP, D-2 XP, E-22 XP, F-2 SP, G-5 SP<br>
| |
| - | (ここに20140919_1を貼付)<br>
| |
| - | (ここに20140919_2を貼付)<br>
| |
| - | <br>
| |
| - | <h5>colony PCR</h5><br>
| |
| - | E-2, E-19 #1~4<br>
| |
| - | E-3, H-5 :No colony<br>
| |
| - | (ここに20140919_3を貼付)<br>
| |
| - | <br>
| |
| - | <h5>ligation → TF</h5><br>
| |
| - | H-5 (G-5 SP + E-22 XP)<br>
| |
| - | H-4 (F-2 SP + E-22 XP)<br>
| |
| - | <br>
| |
| - | <h5>preculture</h5><br>
| |
| - | E-2 E#1, E-19 #1<br>
| |
| - | <br>
| |
| - | <h3>9,20,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>miniprep</h5><br>
| |
| - | E-2, E-19<br>
| |
| - | <br>
| |
| - | <h3>9,22,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa,Nakashima,Takemura,Nakamura,Tara,Yamanaka<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>digestion → gel extraction</h5><br>
| |
| - | A-1 SP, D-2 XP, E-2 ES, E-17 EX, E-22 XP, G-5 SP, J-4 SP<br>
| |
| - | (ここに20140922_1を貼付)<br>
| |
| - | (ここに20140922_2を貼付)<br>
| |
| - | <br>
| |
| - | <h5>colony PCR</h5><br>
| |
| - | H-4 <br>
| |
| - | (ここに20140922_3を貼付)<br>
| |
| - | H-4#1,2,3:OK!<br>
| |
| - | <br>
| |
| - | <h5>ligation - TF</h5><br>
| |
| - | H-5 (G-5 SP + E-22 XP)<br>
| |
| - | J-6 (J-4 SP + E-22 XP)<br>
| |
| - | E-3 (A-1 SP + D-2 XP)<br>
| |
| - | F-1 (E-2 ES + E-17 EX)<br>
| |
| - | <br>
| |
| - | <h3>9,23,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa,Nakashima,Nakamura,Tara,Yamanaka<br>
| |
| - |
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>digestion, gel extraction</h5><br>
| |
| - | A-1CP SP, D-2 XP, E-22 XP, G-5 SP<br>
| |
| - | J-4 SP(stopped)<br>
| |
| - | (IMGP0241)<br>
| |
| - | (IMGP0242)<br>
| |
| - | <br>
| |
| - | <h5>miniprep</h5><br>
| |
| - | H-4(made glycerol stock)<br>
| |
| - | <br>
| |
| - | <h5>colony PCR</h5><br>
| |
| - | J-6<br>
| |
| - | (IMGP0243)<br>
| |
| - | OK!<br>
| |
| - | <br>
| |
| - | <h5>ligation,TF</h5><br>
| |
| - | H-5(G-5 SP+E22 XP)<br>
| |
| - | E-3(A-1 SP+D-2 XP)<br>
| |
| - | <br>
| |
| - | <h3>9,24,2014</h3><br>
| |
| - | <h3>Lab member</h3>Nakashima,Yamanaka,Takemura<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>miniprep</h5><br>
| |
| - | F-1,J-6(made glycerol stock)<br>
| |
| - | E-22,D-6<br>
| |
| - | G-6 did not grow.<br>
| |
| - | We disposed J-6.(dropped on the floor)<br>
| |
| - | <br>
| |
| - | <h5>digestion,gel extraction</h5><br>
| |
| - | A-1 SP,E-2 ES,E-5 ES,E-17 EX,E-18CP EX,E-21 XP,G-5 SP,D-2 XP,F-1 SP,E-19 XP,E-22 XP<br>
| |
| - | (IMGP0244)<br>
| |
| - | (IMGP0245)<br>
| |
| - | <br>
| |
| - | <h5>ligation,TF</h5><br>
| |
| - | J-2(F-1 SP+E-1 XP)<br>
| |
| - | H-1(F-1 SP+E-21 XP)<br>
| |
| - | F-3(E-5 ES+E-17 EX)<br>
| |
| - | F-4(E-2 ES+E-18 EX)<br>
| |
| - | E-3(A-1 SP+D-2 XP)<br>
| |
| - | H-5(G-5 SP+E-22 XP)<br>
| |
| - | <br>
| |
| - | <h3>9,25,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yakashima,Nakamura,Tara,Tsukada,Yamanaka<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>miniprep</h5><br>
| |
| - | A-1CP, J-6<br>
| |
| - | <br>
| |
| - | <h5>digestion,gel extraction</h5><br>
| |
| - | A-1 SP,D-2 XP,E-5 ES,E-17 EX,E-19 XP,E-21 XP,E-22 XP,F-1 SP,G-5 SP<br>
| |
| - | (IMGP0247)<br>
| |
| - | (IMGP0248)<br>
| |
| - | <br>
| |
| - | <h5>ligation,TF</h5><br>
| |
| - | J-2(F-1 SP+E-19 XP)<br>
| |
| - | H-1(F-1 SP+E-21 XP)<br>
| |
| - | F-3(E-5 ES+E-17 EX)<br>
| |
| - | E-3(A-1 SP+D-2 XP)<br>
| |
| - | H-5(G-5 SP+E-22 XP)<br>
| |
| - | <br>
| |
| - | <h5>Making competent cell</h5><br>
| |
| - | <br>
| |
| - | <h5>preculture</h5><br>
| |
| - | G-5,E-5,E-17,<i>E. coli</i> JM109<br>
| |
| - | <br>
| |
| - | <h5>competent cell(made at 0911) check</h5><br>
| |
| - | Ampicillin,Chloramphenicol,non-antibiotics<br>
| |
| - | culture in LB 3ml<br>
| |
| - | LB midium 3ml, as N.C.<br>
| |
| - | culture for 2h.<br>
| |
| - | no TF<br>
| |
| - | <br>
| |
| - | <h5>result of check</h5><br>
| |
| - | Ampicilln,non-antibiotics:become clouded<br>
| |
| - | Chroramphenicol,LB:did not grow<br>
| |
| - | So,we confirmed ampicillin resistant plasmid exists in competent cell and we disposed it.<br>
| |
| - | <br>
| |
| - | <h3>9,26,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa,Nakashima,Tara,Yoshikawa(Fresh),Tsukada<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>miniprep</h5><br>
| |
| - | G-5,E-5,E-17<br>
| |
| - | <br>
| |
| - | <h5>digestion</h5><br>
| |
| - | A-1CP SP,D-2 XP,G-5 EP(strange band appeared,disposed),pSB1C3(A-4) EP,A-1 SP<br>
| |
| - | (IMGP0249)<br>
| |
| - | (IMGP0250)<br>
| |
| - | <br>
| |
| - | <h5>colony PCR</h5><br>
| |
| - | F-3,F-4,H-1,H-5,J-2<br>
| |
| - | (IMGP0251)<br>
| |
| - | (IMGP0253)<br>
| |
| - | OK!<br>
| |
| - | <br>
| |
| - | <h5>ligation,TF</h5><br>
| |
| - | E-3(D-2 XP+A-1SP)<br>
| |
| - | E-3CP(D-2 XP+A-1CP SP)<br>
| |
| - | <br>
| |
| - | <h5>preculture</h5><br>
| |
| - | F-3,F-4,H-1,H-5,J-2#1,G-5<br>
| |
| - | <br>
| |
| - | <h3>9,27,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa,Nakashima<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>TF</h5><br>
| |
| - | E-3(chemical)<br>
| |
| - | <br>
| |
| - | <h5>miniprep</h5><br>
| |
| - | F-3,F-4,H-1,H-5,J-2(made glycerol stock)<br>
| |
| - | G-5<br>
| |
| - | <br>
| |
| - | <h5>TF</h5><br>
| |
| - | E-3CP(electroporation)<br>
| |
| - | <br>
| |
| - | <h3>9,29,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa,Nakashima,Nakamura,Tara,Takemura<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>digestion,gel extraction</h5><br>
| |
| - | A-1CP SP,pSB1C3(A-4) EP,D-2 XP,E-21 XP,G-5 EP,H-5 EP,J-2 SP<br>
| |
| - | (IMGP0256(0929))<br>
| |
| - | (IMGP0257)<br>
| |
| - | <br>
| |
| - | <h5>Gel making</h5><br>
| |
| - | <br>
| |
| - | <h5>ligation</h5><br>
| |
| - | G-5CP(G-5 EP+pSB1C3 EP)<br>
| |
| - | H-5CP(H-5 EP+pSB1C3 EP)<br>
| |
| - | J-5(J-2 SP+E-21 XP)<br>
| |
| - | E-3(A-1CP SP+P-2 XP)<br>
| |
| - | <br>
| |
| - | <h5>preculture</h5><br>
| |
| - | E-5,E-11,E-22<br>
| |
| - | K-1,E-23',Z-1(for assay)<br>
| |
| - | <br>
| |
| - | <h5>PCR</h5><br>
| |
| - | VF2-(ligation product of E-3)-VR
| |
| - | E-3 lig:1uL<br>
| |
| - | VF2:1uL<br>
| |
| - | VR:1uL<br>
| |
| - | Taq:5uL<br>
| |
| - | MilliQ:2uL<br>
| |
| - | Total:10uL<br>
| |
| - | extention:1m12sec<br>
| |
| - | (IMGP0258(0929))<br>
| |
| - | We observed the band which had expected length.<br>
| |
| - | The ligtion must be OK!<br>
| |
| - | <br>
| |
| - | <h3>9,30,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa.Nakamura,Tsukada,Tara,Yamanaka<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>miniprep</h5><br>
| |
| - | E-2,E-5,E-11<br>
| |
| - | <br>
| |
| - | <h5>digestion,gel extraction</h5><br>
| |
| - | We could not find D-2 sample(probably accidentally disposed).<br>
| |
| - | So we stopped it.
| |
| - | <br>
| |
| - | <h5>colony PCR</h5><br>
| |
| - | G-5,H-5,J-5<br>
| |
| - | (IMGP0258(0930))<br>
| |
| - | H-5,J-5#1,2,3,4:OK!<br>
| |
| - | <br>
| |
| - | <h5>Making M9 medium</h5><br>
| |
| - | 1M MgSO4 50ml<br>
| |
| - | 1M CaCl2 10ml<br>
| |
| - | 20% glucose 50ml<br>
| |
| - | <br>
| |
| - | <h5>digestion(cutcheck)</h5><br>
| |
| - | G-5 E,G-5 P,G-5 non-cut<br>
| |
| - | (IMGP0260)<br>
| |
| - | Strange bands appeared.<br>
| |
| - | We decided to read a sequence of this part.<br>
| |
| - | <br>
| |
| - | <h3>10,1,2014</h3><br>
| |
| - | <h3>Lab member</h3>Nakashima,Tara,Nakamura,Takemura<br>
| |
| - | <br>
| |
| - | Contens<br>
| |
| - | <h5>miniprep</h5><br>
| |
| - | H-5CP,G-5CP,J-5(made glycerol stock)<br>
| |
| - | <br>
| |
| - | <h5>sequence order</h5><br>
| |
| - | E-2,E-8,E-9,E-19,F-1,F-3,F-4,G-5,H-1,H-4<br>
| |
| - | H-5(VF-VR,Psigma2F-anti2R),J-2,J-4,J-5,J-6<br>
| |
| - | <br>
| |
| - | <h3>10,2,2014</h3><br>
| |
| - | <h3>Lab member</h3>Nakashima,Nakamura,Tara<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>assay</h5><br>
| |
| - | completely failed<br>
| |
| - | <br>
| |
| - | In M9 medium, <i>Escherichia.coli</i> JM109 did not grow well.(doubling time:1h)<br>
| |
| - | The glycerol stock needed to be put a lot.<br>
| |
| - | <br>
| |
| - | <h5>culture</h5><br>
| |
| - | F-2,F-3,F-4(for M9 check)<br>
| |
| - | <br>
| |
| - | <h5>PCR</h5><br>
| |
| - | G-1(F,R),G-4(F,R),G-5(F,R),H-1(F,R),H-4(F,R),H-5(F,R)<br>
| |
| - | Template:1ng/uL,1uL<br>
| |
| - | <br>
| |
| - | <h5>Making M9 medium</h5><br>
| |
| - | 5*M9 24mL<br>
| |
| - | 20% Glu 1.2mL<br>
| |
| - | MgSO4 240uL<br>
| |
| - | CaCl2 12uL<br>
| |
| - | Amino acid 10mL<br>
| |
| - | mess up to 1L<br>
| |
| - | <br>
| |
| - | <h5>PCR check</h5><br>
| |
| - | (IMPG0261)<br>
| |
| - |
| |
| - | OK!(except G-5)<br>
| |
| - | <br>
| |
| - | <h5>PCR</h5><br>
| |
| - | G-1,G-4,H-1,H-4<br>
| |
| - | extension time:6m30sec<br>
| |
| - | EpCAM nested<br>
| |
| - | 95C 3min-(-95C 30sec-48C 30sec-72C 3min-)*30-72C 5min-4C<br>
| |
| - | <br>
| |
| - | <h5>preculture</h5><br>
| |
| - | E-17,E-18,F-1,F-2,F-3,F-4,Z-1(M9)<br>
| |
| - | E-23'(LB)<br>
| |
| - | <br>
| |
| - | <h3>10,3,2014</h3><br>
| |
| - | <h3>Lab member</h3>Nakashima,Tara,Nakamura<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>PCR clean up and check</h5><br>
| |
| - | (IMGP0262)<br>
| |
| - | G-1,G-4,H-1,H-4:Band in correct position and unexpected position.<br>
| |
| - | EpCAM:No band<br>
| |
| - | <br>
| |
| - | <h5>digestion,gel extraction</h5><br>
| |
| - | (IMGP0263)<br>
| |
| - | (IMGP0264)<br>
| |
| - | G-1deg linear EP,G-4deglinear EP,H-1deg linear EP,H-4deg linear EP(A-4)<br>
| |
| - | (deg:degradation tag)<br>
| |
| - | <br>
| |
| - | <h5>PCR</h5><br>
| |
| - | EpCAM nested<br>
| |
| - | 94C 3min-(-94C 30sec-48C 30sec-72C 3min-)*30-72C 5min-4C
| |
| - | (IMGP0265)<br>
| |
| - | No band<br>
| |
| - | <br>
| |
| - | <h5>gel making</h5><br>
| |
| - | <br>
| |
| - | <h5>ligation,TF</h5><br>
| |
| - | H-1'(H-1'linear EP+pSB1C3 EP)<br>
| |
| - | H-4'(H-4'linear EP+pSB1C3 EP)<br>
| |
| - | G-1'(G-1'linear EP+pSB1C3 EP)<br>
| |
| - | G-4'(G-4'linear EP+pSB1C3 EP)<br>
| |
| - | <br>
| |
| - | <h5>PCR</h5><br>
| |
| - | EpCAM nested<br>
| |
| - | 95C 3min-(-95C 30sec-46C 30sec-72C 3min-)*30-72C 5min-4C
| |
| - | (IMGP0267)<br>
| |
| - | No band
| |
| - | <br>
| |
| - | <h3>10,4,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa<br>
| |
| - | <h5>assay1</h5><br>
| |
| - | F-2 in 20mL culture<br>
| |
| - | When OD600=0.516,we put 10% arabinose 20uL.<br>
| |
| - | F-1 in 20mL culture<br>
| |
| - | When OD600=0.514,we put 10% arabinose 20uL.<br>
| |
| - | Z-1 in 20mL culture<br>
| |
| - | When OD600=0.544,we put 10% arabinose 20uL.<br>
| |
| - | O/N culture<br>
| |
| - | <br>
| |
| - | Culture in 3mL:disposed(OD600 value did not agrees with each other.)<br>
| |
| - | <br>
| |
| - | <h5>colony PCR</h5><br>
| |
| - | H-1deg,H-4deg,G-1eg,G-4deg<br>
| |
| - | We confirmed by fluorescence.<br>
| |
| - | <br>
| |
| - | <h5>preculture</h5><br>
| |
| - | H-1deg,H-4deg#1,G-1eg,G-4deg<br>
| |
| - | <br>
| |
| - | <h3>10,5,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>miniprep</h5><br>
| |
| - | H-1deg,H-4deg#1,G-1eg,G-4deg<br>
| |
| - | <br>
| |
| - | <h5>preculture</h5>(M9)<br>
| |
| - | E-15CP,E-17,E-18CP,I-2,F-1,2,3,4,Z-1<br>
| |
| - | <br>
| |
| - | <h3>10,6,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa,Nakashima,Tara<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>assay1,3</h5><br>
| |
| - | E-15,I-2,F-1,F-2,F-3,F-4,F-17,E-18,Z-1<br>
| |
| - | Measured the OD 600 value.<br>
| |
| - | Except I-2,F-2,the value is more than 1.0.<br>
| |
| - | F-1,F-3,E-17,Z-1(20 fold dilution):Culture in 3mL.
| |
| - | The composition of M9 proved to be wrong,so disposed.<br>
| |
| - | <br>
| |
| - | <h5>Making M9 medium</h5><br>
| |
| - | 5*M9 200mL<br>
| |
| - | 20% Glu 10mL<br>
| |
| - | amino acids 10mL<br>
| |
| - | 1M MgSO4 2mL<br>
| |
| - | 1M CaCl2 100uL<br>
| |
| - | MilliQ 778mL<br>
| |
| - | Total 1000mL<br>
| |
| - | <br>
| |
| - | <h5>result of OD600</h5><br>
| |
| - | F-3(non-Chloramphenicol)<br>
| |
| - | 1h:0.094<br>
| |
| - | 2h:0.086<br>
| |
| - | F-3(Chloramphenicol)
| |
| - | 1h:0.074<br>
| |
| - | 2h:0.073<br>
| |
| - | <br>
| |
| - | <h5>assay1</h5>(tentative)<br>
| |
| - | We measured the fluorescence of F-1,F-2,Z-1(culture in 20mL).<br>
| |
| - | Ex:501nm<br>
| |
| - | F-2:We observed a peak in 511nm.<br>
| |
| - | F-1,Z-1:No peak<br>
| |
| - | <br>
| |
| - | <h5>preculture</h5><br>
| |
| - | F-1,F-2,F-3,F-4,E-15CP,I-2,Z-1,E-17,E-18CP(M9 medium)<br>
| |
| - | E-23'(LB medium)<br>
| |
| - | <br>
| |
| - | <h3>10,7,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa,Nakashima,Tara,Nakamura<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>assay</h5><br>
| |
| - | F-1,F-13,E-17,Z-1,E-15,I-2<br>
| |
| - | culture in M9<br>
| |
| - | except E-15,OD600 is more than 1.0.<br>
| |
| - | E-15:over 0.85<br>
| |
| - | F-2:only 0.3<br>
| |
| - | E-23':over 2.0,culture in LB medium<br>
| |
| - | All samples was cultured in 3 mL.(n=5)<br>
| |
| - | F-1,Z-1:cultured in 20mL(flask)(n=1)<br>
| |
| - | <br>
| |
| - | <h5>OD600 before subculture</h5><br>
| |
| - | E-15:1.113<br>
| |
| - | E-11:0.933<br>
| |
| - | E-18:1.190<br>
| |
| - | E-23':forgot to measure<br>
| |
| - | F-1:0.927<br>
| |
| - | F-2:0.374<br>
| |
| - | F-3:0.878<br>
| |
| - | F-4:0.992<br>
| |
| - | I-2:0.888<br>
| |
| - | Z-1:1.084<br>
| |
| - | <br>
| |
| - | <h5>PCR</h5><br>
| |
| - | J-5(F,R),J-6(F,R)<br>
| |
| - | J-5:OK!<br>
| |
| - | extension time<br>
| |
| - | F:1800+200=2000bp,4min<br>
| |
| - | R:1850+200=2050bp,4min<br>
| |
| - | <br>
| |
| - | <h5>PCR</h5><br>
| |
| - | J-6(F,R)<br>
| |
| - | J-5R<br>
| |
| - | extension time:1min<br>
| |
| - | <br>
| |
| - | <h3>10,8,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshikawa,Nakashima,Tara<br>
| |
| - | <br>
| |
| - | <h3>Contents</h3><br>
| |
| - | <h5>making gel</h5><br>
| |
| - | <br>
| |
| - | <h5>subculture</h5><br>
| |
| - | <i>Escherichia.coli</i>MG1655<br>
| |
| - | 100 fold dilution<br>
| |
| - | 20mL,flask<br>
| |
| - | <br>
| |
| - | <h5>PCR clean up and check</h5><br>
| |
| - | (IMGP0268)<br>
| |
| - | (IMGP0269)<br>
| |
| - | J-6F:low concentration<br>
| |
| - | J-6R:shorter band is OK!<br>
| |
| - | We decided that we made J-5,J-6<br>
| |
| - | as H5deg-positive feedback circuit, and H6deg-positive feedback circuit.<br>
| |
| - | <br>
| |
| - | <h5>sequence order</h5><br>
| |
| - | G-1deg,G-4deg,H-1deg,H-4deg<br>
| |
| - | <br>
| |
| - | <h5>TF</h5>(electroporation)<br>
| |
| - | F-1,F-2,F-3,F-4,E-17,E-18CP,Z-1,I-2,E-15<br>
| |
| - | G-1deg,G-4deg,H-1deg,h-4deg,J-4,J-4<br>
| |
| - | also,2mL culture as recovery.<br>
| |
| - | <br>
| |
| - | <h3>10,9,2014</h3><br>
| |
| - | <h3>Lab member</h3>Yoshiakwa,Nakashima,Tara<br>
| |
| - | <h5>digestion,gel extraction</h5><br>
| |
| - | E-19 XP,E-20 XP,H-1deg SP,H-4deg SP<br>
| |
| - | <br>
| |
| - | <h5>making gel</h5><br>
| |
| - | <br>
| |
| - | <h5>making plate</h5><br>
| |
| - | Chloramphenicol*10<br>
| |
| - | <br>
| |
| - | <h5>ligation</h5><br>
| |
| - | J-5deg(E-19XP+H-1degSP)<br>
| |
| - | J-6deg(E-20XP+H-4degSP)<br>
| |
| - | We did not have competent cell of <i>E.coli</i> MG1655, so put it in freezer.<br>
| |
| - | <br>
| |
| - | <h5>preculture</h5><br>
| |
| - | F-1,E-17,E-15,Z-1,I-2:both in LB medium and M9 medium.<br>
| |
| - | MG1655:LB medium<br>
| |
| - | <br>
| |
| - | <h3>10,10,2014</h3><br>
| |
| - | <h5>assay1,3</h5><br>
| |
| - | I-2,E-15,Z-1,F-1,E-17(made glycerol stock,subculture in 20 fold dilution)<br>
| |
| - | MG1655 did not grow, because we accidentally put antibiotics.
| |
| - | <br>
| |
| - | OD600(O/N culture)<br>
| |
| - | E-15:1.795<br>
| |
| - | E-17:1.781<br>
| |
| - | F-1:1.937<br>
| |
| - | I-2:1.886<br>
| |
| - | Z-1:1.780<br>
| |
| - | <br>
| |
| - | <h5>making M9 medium</h5><br>
| |
| - | <br>
| |
| - | </div>
| |
| | </div> | | </div> |
| | <!-- Modeling --> | | <!-- Modeling --> |
| Line 1,974: |
Line 89: |
| | <!-- Attribution --> | | <!-- Attribution --> |
| | <div id = "Attribution-block" style = "display:none;"> | | <div id = "Attribution-block" style = "display:none;"> |
| - | <div id = "Attribution-1">
| |
| - | <img src="https://static.igem.org/mediawiki/2014/4/44/Sub_attribution.png" class = "contTitle" />
| |
| - | <p>Our activities involved in iGEM were all conducted by undergaduates alone!!<br>Based on fund-raisings and public relations, all team members had a lot of brainstoming, investigations and discussions in order to select project carefully. And we conducted experiments and FINALLY saw results.<br>We also designed and composed all publish tools, and of course, polished all scripts and presentation by ourselves.</p>
| |
| - | <h3>Project</h3>
| |
| - | <p>All we had a lot of brainstorming, investigations and discussion when we decide our projects.<br><b>Yoichi Irie</b>(Team Leader)<br />σ-ReCounter:<br><b>Shunsuke Sumi</b>(idea)<br><b>So Nakashima</b>(idea and comformation)<br><b>Takefumi Yoshikawa</b>(comformation)<br><br>CTCD:<br><b>Masayuki Osawa</b>(conformation)<br><b>Shigetaka Kobari</b>(investigation and conformation)<br><b>Shunsuke Sumi</b>(conformation)<br><b>Senkei Hyo</b>(investigation)<br><b>Yoshihiko Tomofuji</b>(conformation)<br><b>Yshiki Okesaku</b>(investigation)</p>
| |
| - | <h3>Experiment</h3>
| |
| - | <p>Lab. Leader:<br><b>Takefumi Yoshikawa</b>(construction, assay;σ-ReCounter)<br><br>Lab. Members:<br><b>Atsuki Ito</b>(construction)<br><b>Hajime Takemura</b>(construction)<br><b>Keisuke Tsukada</b>(construction)<br><b>Kentaro Tara</b>(construction)<br><b>Kento Nakamura</b>(construction, assay;σ-ReCounter)<br><b>Naruki Yoshikawa</b>(construction)<br><b>Nobuhiro Hiura</b>(construction)<br><b>Shigetaka Kobari</b>(assay;CTCD)<br><b>So Nakashima</b>(construction, Assay;σ-ReCounter)<br><b>Yoshihiko Tomofuji</b>(assay;CTCD)<br><b>Yuto Yamanaka</b>(construction)</p>
| |
| - | <h3>Modeling</h3>
| |
| - | <p><b>Keisuke Tsukada, Kentaro Tara, Manabu Nishiura, Masaki Ono</b></p>
| |
| - | <h3>Web</h3>
| |
| - | <p>Almost all members wrote drafts of our team wiki.<br><b>Cristian David</b>(check our English)<br><b>Hiroki Tsuboi</b>(team website, implementation of our team wiki)</p>
| |
| - | <h3>App</h3>
| |
| - | <p><b>Naruki Yoshikawa</b></p>
| |
| - | <h3>Design</h3>
| |
| - | <p><b>Cristian David</b>(parker design)<br><b>Yoshiki Okesaku</b>(all design, all figure)</p>
| |
| - | <h3>Presentation</h3>
| |
| - | <p><b>Kento Nakamura, Manabu Nishiura, Masato Ishikawa, Yumeno Koga, Yuto Yamanaka</b></p>
| |
| - | <h3>Poster</h3>
| |
| - | <br>
| |
| - | <h3>Public Relation</h3>
| |
| - | <p><b>Ding Yuewen, Keisuke Tsukada</b></p>
| |
| - | <h3>Adviser</h3>
| |
| - | <p><b>Kota Tosimitsu</b></p>
| |
| - | </div>
| |
| - | <div id = "Attribution-2">
| |
| - | <img src="https://static.igem.org/mediawiki/2014/4/44/Sub_Ackno.png" class="contTitle" />
| |
| - | </div>
| |
| - | <div id = "Attribution-3">
| |
| - | <img src="https://static.igem.org/mediawiki/2014/3/38/Sub_sponsors.png" class="contTitle" />
| |
| - | <img src="https://static.igem.org/mediawiki/2014/7/72/Promega.png" class="sponsor">
| |
| - | <p><b>Promega KK.</b>for chamical reagents</p>
| |
| - | <p><b>Teiyukai, Faculty of Engineering, The University of Tokyo</b>for fund</p>
| |
| - | <p><b>Integrated DNA Technologies MBL</b></p>
| |
| - | <img src="https://static.igem.org/mediawiki/2014/3/30/Cosmobio.png" class="sponsor">
| |
| - | <p><b>COSMO BIO Co., Ltd.</b>for fund<br></p>
| |
| - | <img src="https://static.igem.org/mediawiki/2014/1/17/Liveanest.png" class="sponsor">
| |
| - | <p><b>Leave a Nest Co., Ltd.(Hiroyuki Takahashi)</b>for advice for public relations & introduction of Promega KK.</p>
| |
| - | </div>
| |
| | </div> | | </div> |
| | <!-- Human Practice --> | | <!-- Human Practice --> |
| | <div id = "Humanpractice-block" style = "display:none;"> | | <div id = "Humanpractice-block" style = "display:none;"> |
| - | <div id = "Humanpractice-1">
| |
| - | <img src = "https://static.igem.org/mediawiki/2014/1/1c/Sub_introduction.png" class = "contTitle"/>
| |
| - | <p>This year, we set 2 goals in human practice and have made efforts to realize following goals.</p>
| |
| - | <p><b>1.Spreading iGEM in general public.</b></p>
| |
| - | <p><b>2.Activating iGEM in Japan.</b></p>
| |
| - | <p>We set these 2 goals because we want to remove prejudice against gene recombination and synthetic biology, and familiarize people with synthetic biology. By doing these, we think that people can understand synthetic biology better.</p>
| |
| - | <p>We also expect undergraduate students, who will lead next generation of biology, to improve their skills by activating iGEM in Japan.</p> </div>
| |
| - | <div id = "Humanpractice-2">
| |
| - | <img src="https://static.igem.org/mediawiki/2014/4/4b/Sub_to_spread_igem_in_general_public.png" class = "contTitle" />
| |
| - | <h3>"EcoLightsOut"</h3>
| |
| - | <p>We have developed an Android app. to introduce our project. This is a puzzle game which uses the counting system we created. In the project, the state of E. coli is changed by stimulus of signal molecules. In the game, players can stimulate the E. coli by touching it. E. coli are displayed in 3x3 or 5x5 grid shape. When you touch an E. coli in the game, the colors of the E. coli and four adjacent ones are changed. This behavior is analogy of diffusion of signal molecules. The goal is to turn all E. coli green.</p>
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| - | <p>Synthetic biology is not well-known and thought to be unfamiliar to ordinary people. To introduce synthetic biology into public, an easy entrance such as playing game is effective. We hope that players will get interested in synthetic biology by enjoying this game.</p>
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| - | <p>You can download this game from Google Play.</p>
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| - | <img src="https://static.igem.org/mediawiki/2014/8/81/Screenshot1.png" class = "figure" style="width:380px;float:left;" />
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| - | <img src="https://static.igem.org/mediawiki/2014/c/ce/Screenshot2.png" class = "figure" style="width:380px;float:left;" />
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| - | <h3 style="clear:both;">Lectures to general public</h3>
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| - | <h4>School festivals</h4>
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| - | <p>The university of Tokyo has two school festivals per year. The May festival is held in May and the Komaba festival was held in November.We explained iGEM and synthetic biology briefly and introduce our project to audience. We invited other iGEM teams in Japan to May festival. We offered precious opportunities that Japanese iGEM teams meet through May festivals.</p>
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| - | <img src="https://static.igem.org/mediawiki/2014/3/39/Festival1.jpg" class = "figure" style="width:380px;float:left;" />
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| - | <img src="https://static.igem.org/mediawiki/2014/6/6a/Festival2.JPG" class = "figure" style="width:380px;float:left;" />
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| - | <h4 style="clear:both;">Techno-Edge</h4>
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| - | <p>Techno-Edge is the event which the department of technology of university of Tokyo held. The purpose of this event was to appeal department of technology to junior high or high school students.Many laboratories and academic circles such as Robotech took part in this event. iGEM UT-Tokyo also participated in it to appeal synthetic biology.</p>
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| - | <p>Not only high school and junior high, but also primary school students came to our booth and were interested in our explanation.</p>
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| - | <img src="https://static.igem.org/mediawiki/2014/2/27/Technoedge.JPG" class = "figure" />
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| - | <h4>Presentation</h4>
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| - | <p>This year, iGEM Nagahama invited us to the genetics society of Japan, and we participated in it. We made an oral presentation workshop of synthetic biology and took part in poster session. There were many iGEM teams, and we could advertise iGEM in academic world. Moreover, specialists gave advice to us, and we were inspired by professors of synthetic biology.</p>
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| - | <p>We also joined in Japanese Society for Cell Synthesis Research.</p>
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| - | <img src="https://static.igem.org/mediawiki/2014/6/68/Presentation1.jpg" class = "figure" style="width:380px;float:left;" />
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| - | <img src="https://static.igem.org/mediawiki/2014/4/4e/Presentation2.jpg" class = "figure" style="width:380px;float:left;" />
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| - | <h4 style="clear:both;">A cram school</h4>
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| - | <p>We held a seminar in which we explained synthetic biology and iGEM for high school students at a cram school in Komaba.</p>
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| - | </div>
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| - | <div id = "Humanpractice-3">
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| - | <img src="https://static.igem.org/mediawiki/2014/3/34/Sub_to_activate_igem.png" class = "contTitle" />
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| - | <h3>Collaborations</h3>
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| - | <p>We collaborated on modeling with Nagahama.</p>
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| - | <p>Their project aimed cadmium collection using <i>Escherichia coli</i> which has positive chemotaxis for aspartic acid, then we constructed simplified model of chemotaxis
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| - | and simulated behavior of <i>E. coli</i> by using probability and random function.</p>
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| - | <p>Their wiki explained the result of modeling.(→link) We constructed all equations in their simulation including the equation that determines <i>E. coli</i> to choose going straight or turning in probability, and explained equations to them.The figure describing the result of simulation is also made by UT-Tokyo. All code for modeling is here.(→link)</p>
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| - | <h3>Ethics and regulation</h3>
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| - | <p>We thought to confirm whether our project meets ethics, but ethics is very vague, so we decided to research about ethics.</p>
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| - | <h3>iGEM Japan</h3>
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| - | <p>iGEM Japan is an organization which was founded last year for iGEM teams in Japan to cooperate each other.</p>
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| - | <p>In March, iGEM Kyoto held iGEM-Japan West meeting. In this meeting, we shared each team's project and advised each other. This meeting was very useful because we could find our idea's weak point.</p>
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| - | <p>In August, iGEM TMU-Tokyo held iGEM-Japan East meeting, and we made presentations about our projects and criticized each other. Thanks to these meetings, we developed quality of our projects.</p>
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| - | <img src="https://static.igem.org/mediawiki/2014/b/bb/Igemjapan1.jpg" class = "figure" style="width:380px;float:left;" />
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| - | <img src="https://static.igem.org/mediawiki/2014/8/8b/Igemjapan2.jpg" class = "figure" style="width:380px;float:left;" />
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| - | <p style="clear:both;"></p>
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| - | </div>
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| | </div> | | </div> |
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