Team:Colombia/Journal

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<center><b><h1  class="curs1">Journal</h1></b></center>
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[[File:chimii.jpg|200px]]
 
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        Context
 
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It is widely accepted that exposure to stressors increases secretion of glucocorticoids, cortisol, and corticosterone in a wide variety of gnathostome vertebrates (Stephens, 1980) (Greenberg et al, 1987). Research in animals, have demonstrated that allostatic overload of this hormones and other mediators, resulting from chronic stress, causes atrophy of neurons in the hippocampus and prefrontal cortex; brain regions involved in memory, selective attention and executive function; likewise it causes hypertrophy of neurons in the amygdala, the brain region involved in fear and anxiety as well as aggression (McEwen, 2004). This means that prolonged states of stress may lead to impaired performance in daily activities like learning, decision making and ability to remember; additionally, will cause damage on the amygdala which in turn will increase levels of anxiety and aggression.
 
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Other than neurological, chronic stress in vertebrates is tightly related with pathologies upon metabolism, immunity, reproduction, cardiovascular system and cellular proliferation (Harbuz et al, 1992) (Whittier, 1991); Thus, the dysregulation of cortisol and other mediators that form the allostatic system is likely to play a role in many neurophysiological conditions as well as systemic disorders such as diabetes, impaired reproduction and immunosuppression (Rasgon et al, 2005).
 
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Now a days, approximately 26 billion animals, spanning over 10 000 species, are kept on farms and in zoos, conservation breeding centers, research laboratories and households (Manson, 2010). Taking into account that captivity is a well-known acute stressor (Gregory et al, 1996), if proper conditions of the habitat are not reestablished or defined, acute stress may turn into chronic stress, and consequently lead to problems that are particularly undesirable for animals maintained in captivity; including increased abnormal behavior, increased self-injurious behavior, impaired reproduction and immunosuppression (Morgan et al, 2007). Besides, it’s been demonstrated that stressful conditions on cattle elicit changes in muscle gross morphology in direct proportion with the duration of the stressor, thus a negative effect on the meat quality and other products for human consumption (Judge, 1969). 
 
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Likewise, human beings are prone to stressful conditions; Constant exposure to adverse environments like noise and pollution or other lifestyle and social conflicts may cause chronic stress that result, over time, in pathophysiological conditions like atherosclerosis, which can lead to strokes and myocardial infarctions (McEwen, 2007).
 
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Identifying a standardized mechanism that allows an early detection of stress is of high importance in different applications such as animal conservation and human practices such as investigation, medicine, animal breeding and cattle raising. Also, doing the proper characterization of the different types of stress provide insight information about the animal condition and therefore, assist an early diagnosis to prevent acute stress to become chronic.
 
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Nowadays, hormones act as biological markers of stress; In this practices the so called “stress-hormones” such as cortisol in humans and corticosterone in rats are quantified to provide information of stress in animals; Elisa, which is the gold standard for stress determination is highly precise and specific; nevertheless this and most methods that utilize immunoassays are only available for lab practices and involve the manipulation of difficult machinery and specialized procedures that are not available for daily use and which are difficult and expensive to implement in farms, home or other in situ investigations.
 
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We propose a standardized device that’ll detect stress with no need of complex machinery or procedures, but a versatile and easy to use device that can be used by anyone, from a farmer in livestock practices, to yourself at home, to identify if your own pet is held in good conditions and stress-free. The device will sense the presence of one of the “stress hormones”, depending on the specimen used, and compare it to a normal concentration or base line. If the concentration of the hormone sensed is more than usual, the device will send a visual signal to inform the user of the presence of stress. The sensing will be done by a bio-machinery held inside the genetic code of ''Saccharomyces cerevisiae''. Thus, our genetically manufactured yeast will identify if there’s abnormal presence of the hormone and if the threshold established is reached, elicit the alarm signal using m-cherry.
 
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This project that is aimed to a larger population than the methods previously released, can be used for both formal and informal characterization of stress. In previous examination with members of the laboratory of neuroscience of Andes University, they did recognize the asset of the device to objectively characterize behavior with fewer limitations and costs than with ELISA kit. Besides, interviews with veterinarians provide evidence that the animals held in households and other facilities such as animal breeding centers and farms, often require close observation to avoid negative effects associated with stressed animals.
 
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Although we did recognize that identifying stress in animals is of high important in different fields, we propose to  initialize the project with trials on humans mostly because of the vast amount of information on the circadian rhythms and baselines  of cortisol <a href="https://2013.igem.org/Team:Colombia_Uniandes/Implementation ">(read more here)</a>, the main “stress hormone” in humans. Nevertheless, we intent to create a product with different final users, based on personalized needs. This requires a bigger effort as baselines for each specimen shall be established in order to identify the “all or nothing” threshold.
 
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Besides, we propose as future prospects, the possibility to implement various baselines in order to provide a more quantitatively response to the presence of stress, rather than the off-on system originally proposed.
 
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        Glucocorticoid sensor
 
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        <h3 id="parts">Design: Project Parts!</h3>
 
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<h4 id="glucoSensor"> Glucocorticoid sensor</h4>
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[[File:Fotojour.jpg|center|600px]]
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<h5 id="glucoConstruct">Our construct</h5>
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=June 24=
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We plan to use the baker's yeast, ''Saccharomyces cerevisiae'', as a chassis for a plasmid which will contain a chimeric protein used as a transactivating factor in a biosensor with a colored reporter.
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<h5 id="glucoChassis">The Chassis</h5>
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We chose ''S. cerevisiae'' as the chassis because one of the most important parts of our fusion protein, the glucocorticoid receptor hormone binding domain (GCR HBD) is eukaryotic, therefore we wanted an easy to use, easy to grow, eucaryotic vector to express our protein and build our biosensor.
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<h5 id="glucoChimera">The Chimera</h5>
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The glucocorticoid receptor (GCR) from mammals contains three domains necessary for stress hormone related gene transcription, the hormone binding domain (HBD), the DNA binding domain (DNA-BD) and the gene transactivating domain (GTD).
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However, for our construct's performance we used a chimeric protein. Just as the mythological creature made from fused parts from a lion, a goat and a snake, we created a chimeric protein using three domains from different organisms.
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We used the glucocorticoid receptor hormone binding domain (GCR HBD) which came from a rat to recognize our hormones of interest. However, we replaced the other two domains with the herpesvirus gene transactivating domain (HV-GTD) and the yeast's DNA binding domain from GAL4. These two new domains have the advantage of being already used, characterized and being highly efficient. The HV-GTD is a highly efficient transactivating domain, recognized to be several orders of magnitude better than the GCR-GTD.
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Here is the construct:
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[[File:GCtestconstruct1.jpg | thumb | center | upright=3.0 | Glucocorticoid test construct]]
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      How It works
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[[File:howChimi.jpg]]
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<li>Yeast activation with sufficient time prior to any "Stress Test". To do this, take the two part container and rotate the upper part. Then shake it smoothly for a couple of minutes.</li>
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<li>Get the Saliva Sample using the saliva sampler from an animal or human.</li>
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<li>Place the saliva sampler inside the already mixed yeast container. Wait for a few minutes</li>
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<li>Observe the color of the solution in the container. Red colored solution indicates high glugocorticoids concentration (stress behavior).</li>
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The designed device for Chimi manipulation is currently being prototyped using fast prototyping machines at Universidad de los Andes. Benefit-cost ratios for different materials are being studied. We hope to have in short time our first functional device for stress detection, stay tuned!     
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Today was the first day at the lab!! Finally! After finding out the parts we ordered from the registry had been sitting in a locker for the last two weeks and managing to salvage them we re finally up and running.  First thing first, today we are making electrocompetent cells and transforming our first parts. We are using TOP 10 strain to make our transformations. We are transforming mRFP (BBa_K518012), pBAD (BBa_K206000), tetR inverter (BBa_Q04400), amilCp (BBa_K592025), pBAD-CqsA (BBa_K581011), pTet-LuxOD47E (BBa_K218017), pqrr4-GFP (BBa_K581009) and LuxU-CqsS-LuxO (BBa_K581010). We are starting with the Interlab project, so we are transforming those parts as well. For convenience, we have decided to name the Interlab parts IL1 (BBa_I20260, promoter+GFP in 1K3), IL2 (BBa_J23101, promoter), IL3 (E0240, GFP) and IL4 (BBa_J23115, promoter).
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We just realized we are all out of our kanamycin stock. We need to prepare some more tomorrow.
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        Experimental Work
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        <h4 id-"glucoTester">Experimental work  </h4>
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=June 25=
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We have transformants! Well, almost. Nothing grew on the IL4 plate :(. But nothing to worry about, we are just repeating the transformations. We made passes to liquid media of single colonies of all the other parts and we are hoping to do miniprep of all them tomorrow.
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We also cryopreserved all the bacteria we got from the registry. Just made a fresh new Kanamycin stock, so we can now transform tetR inverter.
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=June 26=
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Our first minipreps were unsuccessful and nothing grew on the tetR inverter plate.
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We just realized the tetR inverter part has two other twins in the registry. We are transforming those as well and hope one of them works.  
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        References
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      <h4>References:</h4>
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<li>Greenberg, N., and Wingfield, J. C. (1987). Stress and reproduction: Reciprocal relationships. In ‘‘Hormones and Reproduction in Fishes, Amphibians and Reptiles’’ (D. O. Norris and R. E. Jones, Eds.), pp. 461–503. Plenum, New York.</li>
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<li>Gregory, Lisa. Gross, Timothy. Bolten, Alan. Bjorndal, Karen. Guillette, Louis. (1996) Plasma Corticosterone Concentrations Associated with Acute Captivity Stress in Wild Loggerhead Sea Turtles (Caretta caretta). General and Comparative Endocrinology 104, 312–320</li>
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=June 27=
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<li>Judge, M.D. (1969) Environmental Stress and Meat Quality. Journal of Animal Science, 28: 755-760.</li>
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Today we prepared homemade lysis buffer for our minipreps, but apparently it wasn't very effective. We could not see any DNA after running our miniprerps in a gel.  
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<li>Kirschbaum C, Prussner JC, Stone AA, Federenko I, Gaab J, Lintz D, Schommer N, Hellhammer DH. (1995) Persistent high cortisol responses to repeated psychological stress in a subpopulation of healthy men. Psychosomatic Med 57: 468–474.</li>
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=July 1=
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<li>Manson, Georgia J. (2010) Species differences in responses to captivity: stress, welfare and the comparative method. Trends in Ecology and Evolution. Volume 25, Issue 12: Pages 713–721</li>
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After a long weekend, we are back in business. We have colonies in all the plates with the trnasformations of the tetR inverter! We made passes of all three parts to liquid LB for miniprep tomorrow.  
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<li>McEwen, Bruce S. (2007) Physiology and Neurobiology of Stress and Adaptation: Central Role of the Brain. Physiol Rev 87: 873–904</li>
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=July 2=
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<li>McEwen BS, Chattarji S. (2004) Molecular mechanisms of neuroplasticity and pharmacological implications: the example of tianeptine. Eur Neuropsychopharmacol 14: 497–502.</li>
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We repeated the miniprep of all the parts and finally got it right this time! All molecular weights correspond to what we expected!
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<li>Morgan, Kathleen. Tromborg, Chris. (2007) Sources of stress in captivity. Applied Animal Behaviour Science. Volume 102: 262–302</li>
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=July 3=
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<li>Mirescu C, Gould E. (2006) Stress and adult neurogenesis. Hippocampus16: 233–238</li>
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Now that we have all the parts, we can start building our construct! We are starting with the Interlab parts for now. Today we digested both promoters and GFP , ligated them and transformed them using our electrocompetent TOP 10 E. coli.
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<li>Rasgon NL, Kenna HA. (2005) Insulin resistance in depressive disorders and Alzheimer’s disease: revisiting the missing link hypothesis. Neurobiol Aging 26S: S103–S107</li>
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=July 4=
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All our transformations were unsuccessful. After running the digestions in a gel we realized that the digestion was not complete. We did some positive controls on all the enzymes to verify that they are working properly, but it all seems to be working fine.  
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<li>Sachar EJ, Hellman L, Roffwarg HP, Halpern FS, Fukushima DK, Gallagher TF. (1973)Disrupted 24-hour patterns of cortis8ol secretion in psychotic depression. Arch Gen Psychiarty 28: 19–24</li>
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=July 7=
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<li>Stephens, D. B. (1980). Stress and its measurement in domestic animals: A review of behavioral and physiological studies under field and laboratory conditions. Adv. Vet Sci. Comp. Med. 24, 179–210.</li>
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Today we are measuring fluorescence of the first part of the Interlab Study! We are also trying to begin joining the first two parts of our construct tetR inverter and mRFP. Here is some doodles we used to explain to the new iGemers how the process of digestion and ligation works and the logic behind it.
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=July 8=
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Primers have arrived! We are resuspending our primers and using them to make our first PCRs of the year. We are trying to get pqrr4 and LuxOD47E.
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PCR products have just been confirmed with gel electrophoresis! Finally something is working!
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=July 9=
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Ligation and transformation of the two remaining Interlab parts was a complete failure. We decided to purify the GFP fragment from the digestion and try using this to make the ligation.
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Now that we have the PCR products we are trying to make the following constructs: pqrr4-mRFP, pBAD-LuxOD47E, IL2-GFP, IL4-GFP.
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=July 10=
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Today we digested all the parts with the appropiate enzymes to make the constructs from yesterday. We ligated and trasnformed them. We also decided to start working on tetR inverte-mRFP and tetR inverter-amilCp.
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=July 11=
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No colonies on any of the plates! :(.... We repeated all the transformations from yesterday. Hopefully, something will grow this time.
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=Jul 14=
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We have colonies this time, but they are white. We were expecting red and blue colonies. After verifying the tetR inverter parts we got from the distribution kit, we found out that the size of the digested fragments do not correspond to the 902 bp or the 2070 bp of the inverter and pSB1C3. All three parts look the same. It seems we are not going to be able to use them.
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We are now looking into the possibility of using CI inverter instead of the tetR invertes. We are transforming the two CI invertes from the distrbution kit. Hopefully we'll better luck this time.
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We have not been able to get the two remaining Interlab constructs.
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=July 15=
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We got colonies from CI inverter parts 18B and 18D on 2013 distribution kit! Anddd we got a colony of IL2-3 interlab part! We are making an ON of these parts so we can miniprep tomorrow!
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Also we transformed IL3-4, tetR inverter-amilCp,tetR-RFP on E.coli TOP 10.
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=July 16=
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We got colonies on tetR inverter-RFP and tetR inverter-amilCp transformation but unfortunately all our colonies are white :(…So…something is definitely not working with tetR inverter part.
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Things to do:
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IL4-3 ligation
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Miniprep CI inverter 18B, RFP, AmilCp, pBAD-LuxOD-GFP
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Later that day..
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All the minipreps were good and arabinose 10% w/v solution was prepared to test our pBAD-LuxOD –GFP part. So in order to make the experiment on the fluorimeter tomorrow we left three ON cultures on the shaker.
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=July 17=
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Today we did digestion with SpeI and PstI to our tetR inverter part with buffer 2.1 because we need to confirm that we have the correct bps on the plasmid.
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We also did ligation of: tetR inverter-RFP,tetR inverter-amilCP and IL4-3 , after the ligation we transformed again, so we can have our colonies tomorrow!!! >D
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Amilcp, RFP and GFP digestions were confirmed on gel and tetR inverter digestion is not right, again the digested fragments do not correspond to what they are supposed to. :( We are thinking there is no hope with this part.
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Since we were depressed with tetR inverter, we tried the digestion (SpeI-PstI) with CI inverter (18B and 18D).
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=July 18=
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We made the fluorescence experiment of our pBAD-LuxOD-GFP part and it didn’t work, also we did the digestion and iiiit didn’t work, so we have to make a new plan.
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We have to do the following:
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Repeat pBAD-LuxOD-GFP ligation
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Transform pBAD-LuxOD-GFP ligation and tetR inverter AGAIN
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Miniprep pBAD-LuxOD-GFP and tetR inverter
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Digest pBAD-LuxOD-GFP , CI miniprep and tetR inverter with EcoRI and PstI
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Digest GFP with Xba-Pst
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=July 21=
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There are some parts on the registry that could work for us:
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The first is RBS-tetR-ter-ter-Ptet-RBS-mRFP
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The second is Pcat-RBS-tetR-ter-ter-Ptet-RBS-mRFP
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We thought maybe we could order some primers and amplify only the RBS-tetR-ter-ter-Ptet-RBS-mRFP part of the second construct orrr even better the first one is just right for us in order to add the Pqrr4 promoter amplified by PCR previously!
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Let’s see how it goes! Today we transformed these parts and also if this doesn’t work we will try to make it all from scratch…transforming tetR alone, ter, Ptet and RBS.
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=July 22=
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tetR protein didn’t  work out. Nothing is working out :(
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=July 25=
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Digest: IL2, IL4 and IL2-3
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Gel confirmation
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Ligate : IL2-3, IL4-3
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Transform : IL2-3, IL4-3
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=July 28=
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RBS transformation and RBS-tetR-ter-ter-Ptet-RBS-mRFP in ampicilin BB didnt work out, there are some colonies in doubleTer petri dish and IL2-3 , IL4-3 have NOOO colonies at all.
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We repeated  IL2-3 , IL4-3 transformation in CM BB and RBS-tetR-ter-ter-Ptet-RBS-mRFP and RBS in AMP BB.
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Note: MilliQ Water and eppendorfs must be sterilized!!!
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=July 29=
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We have colonies of:
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RBS-tetR-ter-ter-Ptet-RBS-mRFP
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doubleTer
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RBS
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Ptet
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IL2-3
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We repeated transformation of IL43 and left ON of the rest of the parts so we can miniprep tomorrow.
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=July 30=
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Today we are going to make digestion of all the minipreps:
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RBS-tetR-ter-ter-Ptet-RBS-mRFP (EcoRI-PstI)
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doubleTer (EcoRI-Xba)
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RBS (EcoRI-PstI)
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Ptet (Spe-Pst)
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IL2-3 (Eco-PstI)
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IL4-3(Eco-PstI)
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tetR alone (Eco-Pst)
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And on our gel for today our lovely audience we haveeee:
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RBS-tetR-ter-ter-Ptet-RBS-mRFP (EcoRI-PstI): Many many bands
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doubleTer (EcoRI-Xba): CONFIRMED
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RBS (EcoRI-PstI): CONFIRMED
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Ptet (Spe-Pst): CONFIRMED
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IL2-3 (Eco-Spe): CONFIRMED
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IL4-3(Eco-PstI): CONFIRMED
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tetR alone: CONFIRMED
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We made Ptet-RFP and Ptet-amilCP ligation :)
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=July 31=
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We made ON of IL43, IL23 and pBAD-LuxOD-GFP for fluorimeter
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Ligate RBS-tetR alone and tetR alone-doubleTer
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Transform Ptet-RFP and Ptet-amilCp
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=August 4=
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Miniprep Ptet-RFP, RFP, tetR-dter,amilcp,tet alone
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Digest RBS,RFP,amilcp,tet-dter,tet alone
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Ligate RBS-tetR,RBS-tet-dter
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Transform
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=August 6=
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Up until now we have IL1, IL23 and IL34 parts for interlab and we are waiting for our turn in the fluorimeter. Also we have tetR, tetR-dter,Ptet-RFP and Ptet-amilcp.
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=August 8=
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Digestions…digestions…sad faces…sad faces…we want to go home….we want to go home..
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=August 11=
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We are trying to do the same thing all over and over again and we are going crazy. For real.
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=August 12=
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We made ON of our transformant colonies in RBS-tetR and tetR-dt and also we did our fluorescence experiment today on IL23, pBAD-LuxOD-GFP and IL43. Results will be showed later alligatorrsss!
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=August 13=
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NOT GOOD NEWS....Our negative control (DH5alfa) gave the same results as our beloved parts on IL23, pBAD-LuxOD-GFP and IL43, soooo we will have to go over again.
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We did miniprep today of IL23,IL43, pSB1A3+RFP and tetR-dter.
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Also we digested the linearized plasmid of Amp and also tetR with Eco-Pst
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Digestions:
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IL23 (Eco-Pst)
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IL43 (Eco-Pst)
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tetR (Eco-Spe)
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pSB1A3+RFP (Eco-Pst)
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pSB1A3 ln (Eco-Pst)
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ligations:
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tetR-dter in pSB1A3+RFP
 +
tetR-dter in pSB1A3 ln
 +
 
 +
Transform:
 +
tetR-dter in pSB1A3+RFP
 +
tetR-dter in pSB1A3 ln
 +
RBS-tetR in kan plasmid
 +
pSB1C3+RFP
 +
pSB1K3+RFP
 +
 
 +
We are going to use the pSB1C3+RFP and pSB1K3+RFP plasmids for convinience, so we can see that our part inserted the plasmid by losing its color and appearing white on the petri dish.
 +
 
 +
IL23 and IL43 digestions did not work. All of the others did.
 +
 
 +
=August 14=
 +
 
 +
We did miniprep of pSB1C3+RFP and pSB1K3+RFP.
 +
 
 +
Our minipreps are not going good,its weird.. the concentrations are very very low and we dont know if is the lysis solution. We are going to try working with double of the amount required of lysis solution and also we are going to warm up the water in order to see if the concentration gets higher.
 +
 
 +
=August 15=
 +
 
 +
Today, transmitting  from one computer to another!
 +
 
 +
We did miniprep of pSB1K3+RFP, pSB1K3+RFP and tetR-dTer...but once again minipreps are not working properly.
 +
 
 +
We thought today in a new possible construct, and this time the revoolutionary part is a feedback! it would be like this..
 +
 
 +
[[File:Screen_Shot_2014-10-17_at_10.55.30_PM.png|center|600px]]
 +
 
 +
In this way we can se color much more easier and faster because the reporter is under 2 promoters that activate on the same situation.
 +
 
 +
For this we transformed--> Psid (BBa_I746364) Inducible promoter, activated by psp3 protein
 +
                          RBS+Psp3 (BBa_I746351) Activator of Psid promoter
 +
 
 +
=In our final month=
 +
 
 +
We amplified by PCR RBS-tetR-dter-Ptet-RFP out of the registry part with pCAT.
 +
We amplified Pqrr4 for ligation with the final construct, we still havent succeded by ligating this promoter to the construct.
 +
We obtained pBAD-LUXOD-GFP and made the fluorescence measurements.
 +
We obtained the Psid-Ptet-rbs-RFP-psp3 and we are now workig on a experiment to prove that the system work transforming with tetR protein in a different plasmid under a constitutive promoter and Psid-Ptet-rbs-RFP-psp3 in the same E.coli, by growing this bacteria under different tetracicline concentrations a gradient of color should be obtained.
 +
We obtained rbs-tetR-dter-Pter-RFP
 +
 
 +
Also we are still trying to ligate Pqrr4 promoter to the construct Psid-Ptet-rbs-RFP-psp3 for our final results.

Latest revision as of 03:59, 18 October 2014

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Journal



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Contents

June 24

Today was the first day at the lab!! Finally! After finding out the parts we ordered from the registry had been sitting in a locker for the last two weeks and managing to salvage them we re finally up and running. First thing first, today we are making electrocompetent cells and transforming our first parts. We are using TOP 10 strain to make our transformations. We are transforming mRFP (BBa_K518012), pBAD (BBa_K206000), tetR inverter (BBa_Q04400), amilCp (BBa_K592025), pBAD-CqsA (BBa_K581011), pTet-LuxOD47E (BBa_K218017), pqrr4-GFP (BBa_K581009) and LuxU-CqsS-LuxO (BBa_K581010). We are starting with the Interlab project, so we are transforming those parts as well. For convenience, we have decided to name the Interlab parts IL1 (BBa_I20260, promoter+GFP in 1K3), IL2 (BBa_J23101, promoter), IL3 (E0240, GFP) and IL4 (BBa_J23115, promoter).

We just realized we are all out of our kanamycin stock. We need to prepare some more tomorrow.

June 25

We have transformants! Well, almost. Nothing grew on the IL4 plate :(. But nothing to worry about, we are just repeating the transformations. We made passes to liquid media of single colonies of all the other parts and we are hoping to do miniprep of all them tomorrow.

We also cryopreserved all the bacteria we got from the registry. Just made a fresh new Kanamycin stock, so we can now transform tetR inverter.

June 26

Our first minipreps were unsuccessful and nothing grew on the tetR inverter plate.

We just realized the tetR inverter part has two other twins in the registry. We are transforming those as well and hope one of them works.

June 27

Today we prepared homemade lysis buffer for our minipreps, but apparently it wasn't very effective. We could not see any DNA after running our miniprerps in a gel.

July 1

After a long weekend, we are back in business. We have colonies in all the plates with the trnasformations of the tetR inverter! We made passes of all three parts to liquid LB for miniprep tomorrow.

July 2

We repeated the miniprep of all the parts and finally got it right this time! All molecular weights correspond to what we expected!

July 3

Now that we have all the parts, we can start building our construct! We are starting with the Interlab parts for now. Today we digested both promoters and GFP , ligated them and transformed them using our electrocompetent TOP 10 E. coli.

July 4

All our transformations were unsuccessful. After running the digestions in a gel we realized that the digestion was not complete. We did some positive controls on all the enzymes to verify that they are working properly, but it all seems to be working fine.

July 7

Today we are measuring fluorescence of the first part of the Interlab Study! We are also trying to begin joining the first two parts of our construct tetR inverter and mRFP. Here is some doodles we used to explain to the new iGemers how the process of digestion and ligation works and the logic behind it.

July 8

Primers have arrived! We are resuspending our primers and using them to make our first PCRs of the year. We are trying to get pqrr4 and LuxOD47E.

PCR products have just been confirmed with gel electrophoresis! Finally something is working!

July 9

Ligation and transformation of the two remaining Interlab parts was a complete failure. We decided to purify the GFP fragment from the digestion and try using this to make the ligation. Now that we have the PCR products we are trying to make the following constructs: pqrr4-mRFP, pBAD-LuxOD47E, IL2-GFP, IL4-GFP.

July 10

Today we digested all the parts with the appropiate enzymes to make the constructs from yesterday. We ligated and trasnformed them. We also decided to start working on tetR inverte-mRFP and tetR inverter-amilCp.

July 11

No colonies on any of the plates! :(.... We repeated all the transformations from yesterday. Hopefully, something will grow this time.

Jul 14

We have colonies this time, but they are white. We were expecting red and blue colonies. After verifying the tetR inverter parts we got from the distribution kit, we found out that the size of the digested fragments do not correspond to the 902 bp or the 2070 bp of the inverter and pSB1C3. All three parts look the same. It seems we are not going to be able to use them.

We are now looking into the possibility of using CI inverter instead of the tetR invertes. We are transforming the two CI invertes from the distrbution kit. Hopefully we'll better luck this time.

We have not been able to get the two remaining Interlab constructs.

July 15

We got colonies from CI inverter parts 18B and 18D on 2013 distribution kit! Anddd we got a colony of IL2-3 interlab part! We are making an ON of these parts so we can miniprep tomorrow! Also we transformed IL3-4, tetR inverter-amilCp,tetR-RFP on E.coli TOP 10.

July 16

We got colonies on tetR inverter-RFP and tetR inverter-amilCp transformation but unfortunately all our colonies are white :(…So…something is definitely not working with tetR inverter part. Things to do: IL4-3 ligation Miniprep CI inverter 18B, RFP, AmilCp, pBAD-LuxOD-GFP

Later that day.. All the minipreps were good and arabinose 10% w/v solution was prepared to test our pBAD-LuxOD –GFP part. So in order to make the experiment on the fluorimeter tomorrow we left three ON cultures on the shaker.

July 17

Today we did digestion with SpeI and PstI to our tetR inverter part with buffer 2.1 because we need to confirm that we have the correct bps on the plasmid. We also did ligation of: tetR inverter-RFP,tetR inverter-amilCP and IL4-3 , after the ligation we transformed again, so we can have our colonies tomorrow!!! >D Amilcp, RFP and GFP digestions were confirmed on gel and tetR inverter digestion is not right, again the digested fragments do not correspond to what they are supposed to. :( We are thinking there is no hope with this part. Since we were depressed with tetR inverter, we tried the digestion (SpeI-PstI) with CI inverter (18B and 18D).

July 18

We made the fluorescence experiment of our pBAD-LuxOD-GFP part and it didn’t work, also we did the digestion and iiiit didn’t work, so we have to make a new plan. We have to do the following: Repeat pBAD-LuxOD-GFP ligation Transform pBAD-LuxOD-GFP ligation and tetR inverter AGAIN Miniprep pBAD-LuxOD-GFP and tetR inverter Digest pBAD-LuxOD-GFP , CI miniprep and tetR inverter with EcoRI and PstI Digest GFP with Xba-Pst

July 21

There are some parts on the registry that could work for us: The first is RBS-tetR-ter-ter-Ptet-RBS-mRFP The second is Pcat-RBS-tetR-ter-ter-Ptet-RBS-mRFP We thought maybe we could order some primers and amplify only the RBS-tetR-ter-ter-Ptet-RBS-mRFP part of the second construct orrr even better the first one is just right for us in order to add the Pqrr4 promoter amplified by PCR previously! Let’s see how it goes! Today we transformed these parts and also if this doesn’t work we will try to make it all from scratch…transforming tetR alone, ter, Ptet and RBS.

July 22

tetR protein didn’t work out. Nothing is working out :(

July 25

Digest: IL2, IL4 and IL2-3 Gel confirmation Ligate : IL2-3, IL4-3 Transform : IL2-3, IL4-3

July 28

RBS transformation and RBS-tetR-ter-ter-Ptet-RBS-mRFP in ampicilin BB didnt work out, there are some colonies in doubleTer petri dish and IL2-3 , IL4-3 have NOOO colonies at all.

We repeated IL2-3 , IL4-3 transformation in CM BB and RBS-tetR-ter-ter-Ptet-RBS-mRFP and RBS in AMP BB. Note: MilliQ Water and eppendorfs must be sterilized!!!

July 29

We have colonies of: RBS-tetR-ter-ter-Ptet-RBS-mRFP doubleTer RBS Ptet IL2-3

We repeated transformation of IL43 and left ON of the rest of the parts so we can miniprep tomorrow.

July 30

Today we are going to make digestion of all the minipreps: RBS-tetR-ter-ter-Ptet-RBS-mRFP (EcoRI-PstI) doubleTer (EcoRI-Xba) RBS (EcoRI-PstI) Ptet (Spe-Pst) IL2-3 (Eco-PstI) IL4-3(Eco-PstI) tetR alone (Eco-Pst) And on our gel for today our lovely audience we haveeee: RBS-tetR-ter-ter-Ptet-RBS-mRFP (EcoRI-PstI): Many many bands doubleTer (EcoRI-Xba): CONFIRMED RBS (EcoRI-PstI): CONFIRMED Ptet (Spe-Pst): CONFIRMED IL2-3 (Eco-Spe): CONFIRMED IL4-3(Eco-PstI): CONFIRMED tetR alone: CONFIRMED We made Ptet-RFP and Ptet-amilCP ligation :)

July 31

We made ON of IL43, IL23 and pBAD-LuxOD-GFP for fluorimeter Ligate RBS-tetR alone and tetR alone-doubleTer Transform Ptet-RFP and Ptet-amilCp

August 4

Miniprep Ptet-RFP, RFP, tetR-dter,amilcp,tet alone Digest RBS,RFP,amilcp,tet-dter,tet alone Ligate RBS-tetR,RBS-tet-dter Transform

August 6

Up until now we have IL1, IL23 and IL34 parts for interlab and we are waiting for our turn in the fluorimeter. Also we have tetR, tetR-dter,Ptet-RFP and Ptet-amilcp.

August 8

Digestions…digestions…sad faces…sad faces…we want to go home….we want to go home..

August 11

We are trying to do the same thing all over and over again and we are going crazy. For real.

August 12

We made ON of our transformant colonies in RBS-tetR and tetR-dt and also we did our fluorescence experiment today on IL23, pBAD-LuxOD-GFP and IL43. Results will be showed later alligatorrsss!

August 13

NOT GOOD NEWS....Our negative control (DH5alfa) gave the same results as our beloved parts on IL23, pBAD-LuxOD-GFP and IL43, soooo we will have to go over again. We did miniprep today of IL23,IL43, pSB1A3+RFP and tetR-dter. Also we digested the linearized plasmid of Amp and also tetR with Eco-Pst

Digestions: IL23 (Eco-Pst) IL43 (Eco-Pst) tetR (Eco-Spe) pSB1A3+RFP (Eco-Pst) pSB1A3 ln (Eco-Pst)

ligations: tetR-dter in pSB1A3+RFP tetR-dter in pSB1A3 ln

Transform: tetR-dter in pSB1A3+RFP tetR-dter in pSB1A3 ln RBS-tetR in kan plasmid pSB1C3+RFP pSB1K3+RFP

We are going to use the pSB1C3+RFP and pSB1K3+RFP plasmids for convinience, so we can see that our part inserted the plasmid by losing its color and appearing white on the petri dish.

IL23 and IL43 digestions did not work. All of the others did.

August 14

We did miniprep of pSB1C3+RFP and pSB1K3+RFP.

Our minipreps are not going good,its weird.. the concentrations are very very low and we dont know if is the lysis solution. We are going to try working with double of the amount required of lysis solution and also we are going to warm up the water in order to see if the concentration gets higher.

August 15

Today, transmitting from one computer to another!

We did miniprep of pSB1K3+RFP, pSB1K3+RFP and tetR-dTer...but once again minipreps are not working properly.

We thought today in a new possible construct, and this time the revoolutionary part is a feedback! it would be like this..

Screen Shot 2014-10-17 at 10.55.30 PM.png

In this way we can se color much more easier and faster because the reporter is under 2 promoters that activate on the same situation.

For this we transformed--> Psid (BBa_I746364) Inducible promoter, activated by psp3 protein 

                          RBS+Psp3 (BBa_I746351) Activator of Psid promoter

In our final month

We amplified by PCR RBS-tetR-dter-Ptet-RFP out of the registry part with pCAT. We amplified Pqrr4 for ligation with the final construct, we still havent succeded by ligating this promoter to the construct. We obtained pBAD-LUXOD-GFP and made the fluorescence measurements. We obtained the Psid-Ptet-rbs-RFP-psp3 and we are now workig on a experiment to prove that the system work transforming with tetR protein in a different plasmid under a constitutive promoter and Psid-Ptet-rbs-RFP-psp3 in the same E.coli, by growing this bacteria under different tetracicline concentrations a gradient of color should be obtained. We obtained rbs-tetR-dter-Pter-RFP

Also we are still trying to ligate Pqrr4 promoter to the construct Psid-Ptet-rbs-RFP-psp3 for our final results.

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