Difference between revisions of "Team:UCL/Notebook"
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<li id="item9">Week 9</li> | <li id="item9">Week 9</li> | ||
<li id="item10">Week 10</li> | <li id="item10">Week 10</li> | ||
| − | + | <li id="item11">Week 11</li> | |
| − | <li id="item12"> | + | <li id="item12">Week 12</li> |
| − | + | <!-- <li id="item13">...</li> --> | |
</ul> | </ul> | ||
</div> | </div> | ||
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<a href="#week7"><li id="item7">Week 7</li></a> | <a href="#week7"><li id="item7">Week 7</li></a> | ||
<a href="#week8"><li id="item8">Week 8</li></a> | <a href="#week8"><li id="item8">Week 8</li></a> | ||
| + | <a href="#week9"><li id="item9">Week 9</li></a> | ||
| + | <a href="#week10"><li id="item10">Week 10</li></a> | ||
<a href="#week9"><li id="item9">Week 9</li></a> | <a href="#week9"><li id="item9">Week 9</li></a> | ||
<a href="#week10"><li id="item10">Week 10</li></a> | <a href="#week10"><li id="item10">Week 10</li></a> | ||
| − | + | <a href="#week11"><li id="item11">Week 11</li> | |
| − | + | <a href="#week12"><li id="item12">Week 12</li></a> | |
| − | + | <!-- <li id="item13">...</li> --> | |
</ul> | </ul> | ||
</div> | </div> | ||
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<h3>Tuesday 11<sup>th</sup></h3> | <h3>Tuesday 11<sup>th</sup></h3> | ||
| − | + | <h4>Mind the Gut: Effectors</h4> | |
| − | + | <p> We prepared four mini preps each of antiTNA1 and antiTNA2. Nano drop confirmed the concentration ranging from (65-147)ng/ul. We digested 4 ul of antiTNA from each mini prep with XbaI and PstI. Unfortunately, as the voltage was too high 120 mV, DNA ran out of this 1% gel so we decided to repeat the experiment at lower voltage 100mV in 2% agarose gel. Meanwhile we digested pTAC with S and P and left the digestion overnight. </p> | |
| − | + | <p> No transformed nissle colonies in the plates.We decided to repeat transformation next week </p> | |
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</div> | </div> | ||
<div class="day"> | <div class="day"> | ||
<h3>Thursday 13<sup>th</sup></h3> | <h3>Thursday 13<sup>th</sup></h3> | ||
| − | + | <h4>Mind the Gut: Effectors</h4> | |
| + | <p> We purified CHAT PCR and digested pTac however had very low DNA yield for pTac. Hence, we carried out ligation 1:3 ligation of pTAC and CHAT.</p> | ||
| + | <p>We prepared fresh mini preps of the antiTNA1 and 2 from the previous inoculations as the old mini preps were suspected of contamination. </p> | ||
</div> | </div> | ||
<div class="day"> | <div class="day"> | ||
<h3>Friday 14<sup>th</sup></h3> | <h3>Friday 14<sup>th</sup></h3> | ||
| + | <h4>Mind the Gut: Effectors</h4> | ||
| + | <p>We ran the gel for antiTNAs but only the plasmid backbone was visible. This led us to think either our digestion had not work or because the antiTNAs are only 75bp and 100bp respectively, they were possibly invisible in the gel. </p> | ||
| + | <p>The ligated pTac:Chat was transformed into competent cells and left in sterile desk at room temperature in a dark environment. </p> | ||
</div> | </div> | ||
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<div class="day"> | <div class="day"> | ||
<h3>Monday 17<sup>st</sup></h3> | <h3>Monday 17<sup>st</sup></h3> | ||
| + | <h4>Mind the Gut: Effectors</h4> | ||
| + | <p>We transformed competent Nissle with TPH1 expression cassette plasmid for submission for our art project with talented bio-artmaker <b>Anna Dumitru</b> </p> | ||
</div> | </div> | ||
<div class="day"> | <div class="day"> | ||
<h3>Tuesday 18<sup>nd</sup></h3> | <h3>Tuesday 18<sup>nd</sup></h3> | ||
| − | + | <h4>Mind the Gut: Effectors</h4> | |
| + | <p> We had transformed Nissle with different time periods for heat shock and successfully obtained colonies. As from our experiment, heat shock for 180 seconds works better than for 40 seconds.</p> | ||
| + | <p> We cloned our effectors KAT and GAD in both Ptac and pSB1C3 vectors but antiTNAs only in pSB1C3. We digested, dephosphorylated and ligated the constructs into the designated vectors.</p> | ||
</div> | </div> | ||
<div class="day"> | <div class="day"> | ||
<h3>Wednesday 19<sup>rd</sup></h3> | <h3>Wednesday 19<sup>rd</sup></h3> | ||
| − | + | <h4>Mind the Gut: Effectors</h4> | |
| + | <p> We transformed the effectors with concentrated sample and left them for overnight incubation.</p> | ||
| + | |||
</div> | </div> | ||
<div class="day"> | <div class="day"> | ||
| − | + | <h3>Thursday 20<sup>th</sup></h3> | |
| − | + | <h4>Mind the Gut: Effectors</h4> | |
| + | <p>Our patience and hard work had paid off. We finally had some colonies from the transformation. KAT pSB1C3 was disappointing without any colonies and antiTNA1 had merely one colony </p> | ||
| + | <p> We inoculated the picked colonies in LB media. We picked 4 colonies where possible and left them in shaking incubator for 16 hours. </p> | ||
</div> | </div> | ||
<div class="day"> | <div class="day"> | ||
<h3>Friday 21<sup>st</sup></h3> | <h3>Friday 21<sup>st</sup></h3> | ||
| + | <h4>Mind the Gut: Effectors</h4> | ||
| + | <p> We made mini preps and obtained the DNA concentration from nano drop. </p> | ||
| + | <p> We used 100ng of DNA for diagnostic digestion of each effector. We then ran 2% agarose gel for all the antiTNAs and 1% for the rest.</p> | ||
| + | <p> The gel confirmed the ligation had worked for Pyear TPH and GAD into pSB1C3 and showed slightly unclear results for pTAC-KAT. </p> | ||
| + | <!-- GEL PHOTO HERE--> | ||
| + | <p>AntiTNAs were sadly not visible yet again. However, there was a potential band for antiTNA2 and pSB1C3 plasmid.</p> | ||
| + | <p> We decided to repeat the gel for pTAC-KAT, and antiTNA2 again as they were hopeful but not conclusive. | ||
| + | <!--GEL PHOTO HERE --> | ||
</div> | </div> | ||
</div> | </div> | ||
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<div class="day"> | <div class="day"> | ||
<h3>Monday 24<sup>th</sup></h3> | <h3>Monday 24<sup>th</sup></h3> | ||
| − | + | <h4>Mind the Gut: Effectors</h4> | |
| + | <p> We prepared two separate gels for pTAC-KAT and antiTNA and ran the gel.</p> | ||
| + | <p>The results from the gel were still inconclusive. Only one of the three KAT pSB1C3 digest had double band. The second band had double bands however, the plasmid band was slightly lower as compared to the other bands of same plasmid.</p> | ||
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</div> | </div> | ||
<div class="day"> | <div class="day"> | ||
<h3>Tuesday 25<sup>th</sup></h3> | <h3>Tuesday 25<sup>th</sup></h3> | ||
| − | + | <h4>Mind the Gut: Effectors</h4> | |
| + | <p>We started our work with TPH1 characterisation. We prepared two inoculations of seed culture in LAB media and chrolamphenicol until the growth reached to the optical density of 0.5-0.6. We induced one of the culture with 1mM IPTG and left them overnight in shaking incubator. </p> | ||
| + | <p> Meanwhile, we also prepared 25mM Tris-2 mM EDTA lysis buffer at pH 8.5.</p> | ||
</div> | </div> | ||
<div class="day"> | <div class="day"> | ||
<h3>Wednesday 26<sup>th</sup></h3> | <h3>Wednesday 26<sup>th</sup></h3> | ||
| − | + | <h4>Mind the Gut: Effectors</h4> | |
| + | <p>We centrifuged the whole culture and resuspended with 2ml of lysis buffer.</p> | ||
| + | <p> We lysed the cells by sonication (10 cycles of 10s with 15s break in between) and measured the concentration of TPH1 protein using Bradford assay. Meanwhile, 100mM MES buffer at pH 7 was prepared for making tryptophan solution.</p> | ||
| + | <p>We then made different concentration of TPH1 in 120 ul of tryptophan-Mes solution. </p> | ||
| + | <p> We also prepared solutions with different concentration of 5HTP as a control for the experiment. </p> | ||
| + | <p> Finally we plated the first seven columns of 96 well plates in increasing order of TPH1 concentration and the rest of the columns with increasing 5HTP concentrations which we expected would remain constant.</p> | ||
| + | <p> We expected the fluorescence to increase with increasing TPH1 concentration however, we got mixed result. We had performed the assay with excitation at 320 wavelength and emission at 420 nm. However, the paper we had based our assay on advised execution at 300nm and to measure emission at 330 nm, so we decided to repeat the process with these changes. <p> | ||
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</div> | </div> | ||
<div class="day"> | <div class="day"> | ||
<h3>Thursday 27<sup>th</sup></h3> | <h3>Thursday 27<sup>th</sup></h3> | ||
| − | + | <h4>Mind the Gut: Effectors</h4> | |
| + | <p> Prepared the induced and uninduced sample as on Tuesday.</p> | ||
</div> | </div> | ||
<div class="day"> | <div class="day"> | ||
<h3>Friday 28<sup>th</sup></h3> | <h3>Friday 28<sup>th</sup></h3> | ||
| − | + | <h4>Mind the Gut: Effectors</h4> | |
| + | <p> We centrifuged and lysed our cells by sonication. </p> | ||
| + | <p> We performed the assay according to the protocol with only changing the excitation to 300nm and emission at 330 nm in fluorescense plate counter. We had good data, as the fluoresense increased with increasing concentration of TPH1 and remained constant for control sample. Yayyy! </p> | ||
</div> | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
| − | + | ||
| + | <div class="about-item12 hide"> | ||
| + | <div class="notebookweek" id="week12"> | ||
| − | < | + | <h2>Week 12</h2>(31<sup>st</sup> Aug– 6<sup>th</sup> Sep)</h2> |
| − | + | <div class="day"> | |
| + | <h3>Monday 31<sup>st</sup></h3> | ||
| + | <p style="color:red"> BANK HOLIDAY </p></div> | ||
| + | <div class="day"> | ||
| + | <h3>Tuesday 1<sup>st</sup></h3> | ||
| + | <h4>Mind the Gut: Effectors</h4> | ||
| + | <p> We cloned KAT AND GAD into pTAC and Pyear-TPH1 into pSBIC3. We then transformed them into competent cells and left them to grow overnight.</p> | ||
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| + | </div> | ||
| + | <div class="day"> | ||
| + | <h3>Wednesday 3<sup>rd</sup></h3> | ||
| + | <h4>Mind the Gut: Effectors</h4> | ||
| + | <p> No colonies in any plate. We left them for longer to incubate as some cultures had previously shown colonies on second day of incubation. </p> | ||
| + | </div | ||
| − | </div></div> | + | <div class="day"> |
| + | <h3>Thursday 4<sup>th</sup></h3> | ||
| + | |||
| + | </div> | ||
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| + | <div class="day"> | ||
| + | <h3>Friday 5<sup>th</sup></h3> | ||
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| + | </div> | ||
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| + | </div> </div> | ||
| + | <!-- | ||
<div class="about-item13 hide"> | <div class="about-item13 hide"> | ||
Latest revision as of 00:37, 19 September 2015
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