Team:Colombia/Journal

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<h2>Journal</h2>
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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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<h5 id="glucoConstruct">Our construct</h5>
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=='''CONTENIDO'''==
<p align="justify">
<p align="justify">
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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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Lalalala.
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<h5 id="glucoChassis">The Chassis</h5>
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===='''CONTENIDO 2'''====
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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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Lalalalla
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<h5 id="glucoChimera">The Chimera</h5>
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<ol>
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<li>ej1</li>
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<li>ej2</li>
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<li>ej3</li>
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</ol>
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==== '''15th June 2013''' ====
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We picked the transformant colonies.
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===='''18th June 2013'''====
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<p>We performed miniprep procedures with the GenElute HP Plasmid Miniprep kit.</p>
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<p>This are the overall steps:</p>
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<ol>
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<li>Harvest cells.</li>
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<li>Resuspend cells.</li>
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<li>Cell lysis.</li>
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<li>Neutralization.</li>
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Spin method:
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<li>Prepare column.</li>
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<li>Load cleared lysate.</li>
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<li>Wash column with wash solution 1.</li>
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<li>Was column with wash solution 2.</li>
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<li>Centrifuge.</li>
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<li>Elute DNA.</li>
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</ol>
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We did a confirmation Gel 100 V x 30 min. -> It showed 1 bond in the first two wells corresponding to Nal1 and Nal2. We succesfully extracted the Nal plasmids!
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===='''June 21st, 2013'''====
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Harju et al., “Bust n’ Grab” Protocol for Yeast Genomic DNA Extraction
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<ol>
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<li>5 mL of overnight culture of ''S. cerevisiae'' (in BHI medium) were centrifuged at 8500 rpm for 5 min. Discard de supernatant.</li>
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<li>500 µL of Harju lysis buffer were added to each tube.</li>
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<li>Place 2 min at -20 °C, 1 min in water bath at 90 °C and repeat.</li>
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<li>Vortex 30 s.</li>
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<li>Add 500 µL of chloroform, vortex 2 min and centrifuge 3 min at 8500 rpm.</li>
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<li>Transfer the upper aqueous phase to a tube with 800 µL of chilled 100% ethanol and mix by inversion.</li>
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<li>Incubate for 5 min at room temperature or at 30 °C.</li>
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<li>Centrifuge for 5 min, 8500 rpm, and discard supernatant.</li>
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<li>Wash the pellet with 500 µL of ethanol (100%) by vortex. Repeat step 9.</li>
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<li>Dry pellets at room temperature or at 60 °C.</li>
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<li>Resuspend in 40 µL miliQ water.</li>
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</ol>
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===='''June 26, 2013'''====
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<ul>
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<li>We made competent yeast following the procedure mentioned before.</li>
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<li>We also made our first PCRs! We used primers 6 & 1 (A) and 34 & 9 (B) to extract VP16 and GCR from the Nal1 plasmid.</li>
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<li>Confirmation gel (2013-06-26 19 hr 16 min.jpg & 2013-06-26 19hr 15 min.jpg) with wells:
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<ol>
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<li>Ladder</li>
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<li>PCR A</li>
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<li>PCR B</li>
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<li>Miniprep for Nal. 1</li></ol></li>
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</ul>
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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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A = VP16
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<br>B = GCR
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</p>
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[[File:Construct.jpg|400px|thumb|center]]
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===='''June 27, 2013'''====
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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 repeated the PCR for A and used lambda phage DNA for carrier DNA.
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We also tried extracting yeast genome using a modified Harju “Bust n’ Grab” protocol using three parallel methods:
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<ul>
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<li>Method “H” used the regular lysis buffer.</li>
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<li>Method “C” used the following lysis buffer: 3% Triton, 100 mM LiCl, 10 mM Tris-HCl, 1 mM EDTA, 100 mM NaAc.</li>
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<li>Method “O” used the following lysis buffer: 2% triton, 1& SDS, 10 mM tris-HCl, 1 mM EDTA, 100 mM LiCl.</li>
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</ul>
</p>
</p>
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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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Conformation gel was run with wells:
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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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<ol>
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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>Ladder</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>PCR A (repeated)</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>Carrier lambda PCR</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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<li>Method C</li>
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<li>Method H</li>
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<li>Method O</li>
</ol>
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The genome extraction still isn't working! :(
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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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<p align="justify">
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We then ran a fusion PCR with GAL4 and VP16 (A and B). These were the PCR conditions:
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<ul>
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<li>1st PCR --> 2-step PCR
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<br>Cycle steps
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<ol>
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<li>Initial denaturation (98 °C, 30 s)</li>
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<li>15 cycles (98 °C, 10 s; 72 °C, 35 s)</li>
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<li>Final extension (72 °C, 5 min)</li>
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<li>Hold (4 °C, indefinite time)</li>
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</ol></li>
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<li>2nd PCR --> 2-step PCR (add primers)
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<br>Cycle steps
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<li>Initial denaturation (98 °C, 30 s)</li>
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<li>35 cycles (98 °C, 10 s; 72 °C, 35 s)</li>
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<li>Final extension (72 °C, 10 min)</li>
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        Experimental Work
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<li>Hold (4 °C, indefinite time)</li>
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        <h4 id-"glucoTester">Experimental work  </h4>
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===='''July 2nd, 2013'''====
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Still trying to successfully extract the yeast genome! This time we tried an alternate method where we used two different solutions to break the cell wall:
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<ul>
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<li>Solution I: Glucose 50 mM, EDTA pH 8 10 mM, Tris-HCl pH 8 25 mM (esterilized) + Zymolyase</li>
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<li>Solution II: NaOH 0.2 N, SPS 1%</li>
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The rest of the protocol was taken from GenElute DNA Kit from Sigma-Aldrich.
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</ul>
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===='''July 3rd, 2013'''====
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The genome extraction was better, but it's mostly degraded DNA! We still have to improve the protocol.
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===='''July 5th, 2013'''====
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We're still improving our genome extraction protocol. This time we're trying 4 variations to break the cell wall
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<br>We're varying the incubation of both solutions, the one that comes with the kit (Proteinase K + Lysis buffer) and the zymolyase solution we previously used. The four variations were as follows.
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<ul>
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<li>A: Zymolyase for ½ h at 37 °C</li>
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<li>B: Zymolyase for ½ h at 37 °C, then protease K + lysis T ½ h at 55 °C</li>
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<li>C: Zymolyase for ½ h at 37 °C, then protease K + lysis T ½ h at 37 °C</li>
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<li>D: Regular GenElute Genome extraction protocol</li>
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</ul>
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The rest of the steps were done following the instructions from the GenElute Genome extraction protocol.
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<br>We ran a gel in this order: WM, A, B, C, D.
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<br>Both B and C gave results, with C giving better yields! We're keeping the C protocol and we're happy we can start extracting parts from the yeast genome!
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[[File:GENOMES.jpg|400px|thumb|center]]
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<br>We performed PCRs for BAP2, GAL4, yeast terminator and pGAL1. These were the conditions:
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<br>PCR fusion 2 steps*, with process 3 at 72 °C for 45 s
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<ol>*
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<li>98 °C, 1 min</li>
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<li>98 °C, 10 s</li>
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<li>72 °C, 45 s</li>
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        References
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Steps 2 and 3 x 35
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<li>72 °C, 10 min</li>
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<li>4 °C</li>
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      <h4>References:</h4>
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<ol>
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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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===='''July 9th, 2013'''====
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Today we ran PCRs for pGAL1 and mCherry. Now that we have several parts, we must have a clear understanding of our notation!
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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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===='''July 18th, 2013'''====
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Today we ran PCRs for the following fusions: F1 = TER-GCR, with primers 31 & 35; F2 = TER – mCherry, with primers 31 & 33. We also amplified E = pGAL1, using primers 13 & 15.
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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 19th, 2013'''====
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Today we amplified F2 that we obtained yesterday.
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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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===='''July 22nd, 2013'''====
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Today we tried to fuse C-D using primers 19 and 5, TER-GCR using primers 31 and 35 and TER-mCherry using primers 31 and 33. After 15 PCR cycles, we added the primers.
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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 24th, 2013'''====
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Today we extracted the terminator from the yeast genome (F1 and F2) and pGAL1 from miniprep (E). We also did PCRs to fuse again our big parts: pBAP2 – GAL4 – VP16 – GCR (CD-AB) (primers 19 & 34) and pGAL1 – mCherry (E-G) (primers 32 & 15).
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<br>At the moment, this is our progress!:
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[[File:Progress_chart.jpg|400px|thumb|center]]
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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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===='''August 5th, 2013'''====
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Things are advancing at a fast pace! Today we did miniprep to obtain PUC19!
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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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===='''August 6th, 2013'''====
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After doing a confirmation gel, C and CD are no longer with us! So we need to repeat them!
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<li>Mirescu C, Gould E. (2006) Stress and adult neurogenesis. Hippocampus16: 233–238</li>
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===='''August 16th, 2013'''====
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Still no C nor D, so we did PCRs again with different methods to obtain C1, C2, D1 and D2.
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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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===='''August 17th,2013'''====
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We're now starting with the reporter construct's PCRs. We got the synthetised parts (E2, E3, E4 and F (the terminator)) which means we need to start fusing them. First off, we'll fuse the terminator with the reporter, since it's common to all reporters.
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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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===='''August 19th,2013'''====
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Today we'll perform the fusion between FG and the four Es.  
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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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===='''August 20th,2013'''====
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The fusion didn't work out, so we reamplified FG and tried again.
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</ol>
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===='''August 30th, 2013'''====
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We finally have the E*GF fusions complete!
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We did the digestion for the E*GF parts and the pSB1C3 plasmid as follows:
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<ul>
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<li>Buffer 5x: 5 uL</li>
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<li>SpeI: 1 uL</li>
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<li>XbaI: 1 uL</li>
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<li>DNA: 10 uL </li>
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<li>Water: 33 uL </li>
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</ul>
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They were placed for two hours at 37°C and then were inactivated at 80°C for 20 minutes. Then we added alkaline phosphatase for 1 hour at 37°C.
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      </div>
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Then, we did the ligation protocol as follows:
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    </div>
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<ul>
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  </div>
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<li>Buffer 5x: 2 uL</li>
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</div>
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<li>plasmid: 3 uL</li>
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    </div>
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<li>insert: 9 uL</li>
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  </div>
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<li>ligase: 1 uL</li>
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</div>
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<li>Water: 5 uL</li>
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</ul>
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We then transformed them. Tomorrow we'll confirm the transformation by miniprep kit.
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===='''August 31st'''====
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After transforming them we got these minipreps
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[[File:GELminipreps.jpg|400px|thumb|center]]
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Here we can see all four E*GF parts (the four to the left of the weight marker), some with more than one plasmid conformation.
 +
===='''September 2nd, 2013'''====
 +
We contransformed the E*GF parts with the NAL plasmid in order to validate their function. Once again we did electrocompetent cells and transformed immediately.
 +
===='''September 3rd, 2013'''====
 +
We did the PCR confirmation for the parts. The bands aren't very bright but it's enough for us! We'll run the validation experiments tomorrow!
 +
Wells are as follows: E1FG - E1FG(2) - E2FG - E2FG(2) - Weight marker - E3FG - E3FG(2) - E4FG - E4FG(2).
 +
Something strange happened with E3FG(2)
 +
[[File:GEL7.jpg|400px|thumb|center]]
 +
===='''September 4th, 2013'''====
 +
Everything is ready for the validation experiments. We're both nervous and excited!
 +
We inoculated cells with dexamethasone. The original syringe had a concentration of 8mg/2mL. We obtained a total initial volume of 3.189*10^-2 µL, so we had to dilute first the dexamethasone: 9900 µL water + 100 µL dexamethasone.
 +
Induction experiment ON: 5 mL LB + 20 µL ampicillin + 20 µL kanamycin + 10 µL inoculum + 32 µL dexamethasone (1:1000) (inoculate ON).
 +
We ran a gel for several PCRs: F-H-P-E1GF2-MP-E2GF-E3GF1-E3GF2-E4GF- Everything was succesful except E1GF2.
 +
Then we digested the PCRs with 5 µL Buffer CutSmart + 15 µL PCR + 1 µL XbaI + 1 µL SpeI + 28 µL water (for each PCR)
 +
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=='''COTENIDO OTRO MES'''==
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Lalalala
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Revision as of 16:57, 19 June 2014

Journal

Contents

CONTENIDO

Lalalala.

CONTENIDO 2

Lalalalla

  1. ej1
  2. ej2
  3. ej3


15th June 2013

We picked the transformant colonies.

18th June 2013

We performed miniprep procedures with the GenElute HP Plasmid Miniprep kit.

This are the overall steps:

  1. Harvest cells.
  2. Resuspend cells.
  3. Cell lysis.
  4. Neutralization.

    5. Spin method:

  5. Prepare column.
  6. Load cleared lysate.
  7. Wash column with wash solution 1.
  8. Was column with wash solution 2.
  9. Centrifuge.
  10. Elute DNA.

We did a confirmation Gel 100 V x 30 min. -> It showed 1 bond in the first two wells corresponding to Nal1 and Nal2. We succesfully extracted the Nal plasmids!

June 21st, 2013

Harju et al., “Bust n’ Grab” Protocol for Yeast Genomic DNA Extraction

  1. 5 mL of overnight culture of S. cerevisiae (in BHI medium) were centrifuged at 8500 rpm for 5 min. Discard de supernatant.
  2. 500 µL of Harju lysis buffer were added to each tube.
  3. Place 2 min at -20 °C, 1 min in water bath at 90 °C and repeat.
  4. Vortex 30 s.
  5. Add 500 µL of chloroform, vortex 2 min and centrifuge 3 min at 8500 rpm.
  6. Transfer the upper aqueous phase to a tube with 800 µL of chilled 100% ethanol and mix by inversion.
  7. Incubate for 5 min at room temperature or at 30 °C.
  8. Centrifuge for 5 min, 8500 rpm, and discard supernatant.
  9. Wash the pellet with 500 µL of ethanol (100%) by vortex. Repeat step 9.
  10. Dry pellets at room temperature or at 60 °C.
  11. Resuspend in 40 µL miliQ water.

June 26, 2013

  • We made competent yeast following the procedure mentioned before.
  • We also made our first PCRs! We used primers 6 & 1 (A) and 34 & 9 (B) to extract VP16 and GCR from the Nal1 plasmid.
  • Confirmation gel (2013-06-26 19 hr 16 min.jpg & 2013-06-26 19hr 15 min.jpg) with wells:
    1. Ladder
    2. PCR A
    3. PCR B
    4. Miniprep for Nal. 1

A = VP16
B = GCR

File:Construct.jpg
400px

June 27, 2013

We repeated the PCR for A and used lambda phage DNA for carrier DNA. We also tried extracting yeast genome using a modified Harju “Bust n’ Grab” protocol using three parallel methods:

  • Method “H” used the regular lysis buffer.
  • Method “C” used the following lysis buffer: 3% Triton, 100 mM LiCl, 10 mM Tris-HCl, 1 mM EDTA, 100 mM NaAc.
  • Method “O” used the following lysis buffer: 2% triton, 1& SDS, 10 mM tris-HCl, 1 mM EDTA, 100 mM LiCl.

Conformation gel was run with wells:

  1. Ladder
  2. PCR A (repeated)
  3. Carrier lambda PCR
  4. Method C
  5. Method H
  6. Method O

The genome extraction still isn't working! :(

We then ran a fusion PCR with GAL4 and VP16 (A and B). These were the PCR conditions:

  • 1st PCR --> 2-step PCR
    Cycle steps
    1. Initial denaturation (98 °C, 30 s)
    2. 15 cycles (98 °C, 10 s; 72 °C, 35 s)
    3. Final extension (72 °C, 5 min)
    4. Hold (4 °C, indefinite time)
  • 2nd PCR --> 2-step PCR (add primers)
    Cycle steps
    1. Initial denaturation (98 °C, 30 s)
    2. 35 cycles (98 °C, 10 s; 72 °C, 35 s)
    3. Final extension (72 °C, 10 min)
    4. Hold (4 °C, indefinite time)

    • July 2nd, 2013

    Still trying to successfully extract the yeast genome! This time we tried an alternate method where we used two different solutions to break the cell wall:

    • Solution I: Glucose 50 mM, EDTA pH 8 10 mM, Tris-HCl pH 8 25 mM (esterilized) + Zymolyase
    • Solution II: NaOH 0.2 N, SPS 1%

      • The rest of the protocol was taken from GenElute DNA Kit from Sigma-Aldrich.

    July 3rd, 2013

    The genome extraction was better, but it's mostly degraded DNA! We still have to improve the protocol.

    July 5th, 2013

    We're still improving our genome extraction protocol. This time we're trying 4 variations to break the cell wall
    We're varying the incubation of both solutions, the one that comes with the kit (Proteinase K + Lysis buffer) and the zymolyase solution we previously used. The four variations were as follows.

    • A: Zymolyase for ½ h at 37 °C
    • B: Zymolyase for ½ h at 37 °C, then protease K + lysis T ½ h at 55 °C
    • C: Zymolyase for ½ h at 37 °C, then protease K + lysis T ½ h at 37 °C
    • D: Regular GenElute Genome extraction protocol

    The rest of the steps were done following the instructions from the GenElute Genome extraction protocol.


    We ran a gel in this order: WM, A, B, C, D.
    Both B and C gave results, with C giving better yields! We're keeping the C protocol and we're happy we can start extracting parts from the yeast genome!

    File:GENOMES.jpg
    400px



    We performed PCRs for BAP2, GAL4, yeast terminator and pGAL1. These were the conditions:
    PCR fusion 2 steps*, with process 3 at 72 °C for 45 s

      *
    1. 98 °C, 1 min
    2. 98 °C, 10 s
    3. 72 °C, 45 s

      4. Steps 2 and 3 x 35

    4. 72 °C, 10 min
    5. 4 °C

    July 9th, 2013

    Today we ran PCRs for pGAL1 and mCherry. Now that we have several parts, we must have a clear understanding of our notation!

    July 18th, 2013

    Today we ran PCRs for the following fusions: F1 = TER-GCR, with primers 31 & 35; F2 = TER – mCherry, with primers 31 & 33. We also amplified E = pGAL1, using primers 13 & 15.

    July 19th, 2013

    Today we amplified F2 that we obtained yesterday.

    July 22nd, 2013

    Today we tried to fuse C-D using primers 19 and 5, TER-GCR using primers 31 and 35 and TER-mCherry using primers 31 and 33. After 15 PCR cycles, we added the primers.

    July 24th, 2013

    Today we extracted the terminator from the yeast genome (F1 and F2) and pGAL1 from miniprep (E). We also did PCRs to fuse again our big parts: pBAP2 – GAL4 – VP16 – GCR (CD-AB) (primers 19 & 34) and pGAL1 – mCherry (E-G) (primers 32 & 15).
    At the moment, this is our progress!:

    File:Progress chart.jpg
    400px

    August 5th, 2013

    Things are advancing at a fast pace! Today we did miniprep to obtain PUC19!

    August 6th, 2013

    After doing a confirmation gel, C and CD are no longer with us! So we need to repeat them!

    August 16th, 2013

    Still no C nor D, so we did PCRs again with different methods to obtain C1, C2, D1 and D2.

    August 17th,2013

    We're now starting with the reporter construct's PCRs. We got the synthetised parts (E2, E3, E4 and F (the terminator)) which means we need to start fusing them. First off, we'll fuse the terminator with the reporter, since it's common to all reporters.

    August 19th,2013

    Today we'll perform the fusion between FG and the four Es.

    August 20th,2013

    The fusion didn't work out, so we reamplified FG and tried again.

    August 30th, 2013

    We finally have the E*GF fusions complete! We did the digestion for the E*GF parts and the pSB1C3 plasmid as follows:

    • Buffer 5x: 5 uL
    • SpeI: 1 uL
    • XbaI: 1 uL
    • DNA: 10 uL
    • Water: 33 uL

    They were placed for two hours at 37°C and then were inactivated at 80°C for 20 minutes. Then we added alkaline phosphatase for 1 hour at 37°C.

    Then, we did the ligation protocol as follows:

    • Buffer 5x: 2 uL
    • plasmid: 3 uL
    • insert: 9 uL
    • ligase: 1 uL
    • Water: 5 uL

    We then transformed them. Tomorrow we'll confirm the transformation by miniprep kit.

    August 31st

    After transforming them we got these minipreps

    File:GELminipreps.jpg
    400px

    Here we can see all four E*GF parts (the four to the left of the weight marker), some with more than one plasmid conformation.

    September 2nd, 2013

    We contransformed the E*GF parts with the NAL plasmid in order to validate their function. Once again we did electrocompetent cells and transformed immediately.

    September 3rd, 2013

    We did the PCR confirmation for the parts. The bands aren't very bright but it's enough for us! We'll run the validation experiments tomorrow!

    Wells are as follows: E1FG - E1FG(2) - E2FG - E2FG(2) - Weight marker - E3FG - E3FG(2) - E4FG - E4FG(2). Something strange happened with E3FG(2)

    File:GEL7.jpg
    400px

    September 4th, 2013

    Everything is ready for the validation experiments. We're both nervous and excited!

    We inoculated cells with dexamethasone. The original syringe had a concentration of 8mg/2mL. We obtained a total initial volume of 3.189*10^-2 µL, so we had to dilute first the dexamethasone: 9900 µL water + 100 µL dexamethasone.

    Induction experiment ON: 5 mL LB + 20 µL ampicillin + 20 µL kanamycin + 10 µL inoculum + 32 µL dexamethasone (1:1000) (inoculate ON).

    We ran a gel for several PCRs: F-H-P-E1GF2-MP-E2GF-E3GF1-E3GF2-E4GF- Everything was succesful except E1GF2.

    Then we digested the PCRs with 5 µL Buffer CutSmart + 15 µL PCR + 1 µL XbaI + 1 µL SpeI + 28 µL water (for each PCR)


COTENIDO OTRO MES

Lalalala

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