Team:Cornell/testing/notebook/wetlab/4
From 2012.igem.org
(Difference between revisions)
Line 271: | Line 271: | ||
<div class="nine columns"> | <div class="nine columns"> | ||
<h3>Week 4</h3> | <h3>Week 4</h3> | ||
- | + | Conjugating nah into Shewy: A gel of last week's colony PCRs hinted that one of the colonies had the nah operon in it! Excitement! Liquid cultures were made, and there was just enough DNA after miniprepping to submit for sequencing. We will collectively hold our breaths and wait for positive results.<br><br> | |
+ | This week, we were able to begin characterizing the current response to arsenic of S20 (JG700 + p14k; see strain list). Additionally, we submitted physical DNA for six BioBrick parts to the parts registry. | ||
<a href="#" class="technical-desc" for="#technical-desc3" style="display:block;margin-top:20px;">Daily Details</a> | <a href="#" class="technical-desc" for="#technical-desc3" style="display:block;margin-top:20px;">Daily Details</a> | ||
<div class="hide-me panel" style="background:white;margin-top: 20px;" id="technical-desc3"> | <div class="hide-me panel" style="background:white;margin-top: 20px;" id="technical-desc3"> | ||
<h6>Daily Details:</h6> | <h6>Daily Details:</h6> | ||
- | + | <b>September 23rd</b> | |
+ | <br><br> | ||
+ | <b>Conjugating nah into Shewy:</b> | ||
+ | Caleb and Tina ran a gel of the colony PCRs and positive control PCR. Swati also started cultures of our arsenic-sensitive strain, as well as control strains, for a plate testing our arsenic reporters. | ||
+ | <br><br> | ||
+ | <b>September 24th</b> | ||
+ | <br><br> | ||
+ | <b>Conjugating nah into Shewy:</b> | ||
+ | Caleb inoculated 30 mL LB+DAP+Cm with the 24 hour liquid culture of of colony N. | ||
+ | <br><br> | ||
+ | <b>September 25th</b> | ||
+ | <br><br> | ||
+ | <b>Fluorescence tests:</b> Swati set up a conjugation of SAL2_mRFP into JG700. Also, sequencing came back good so once we have this part in Shewanella we can start testing our salicylate sensor. Jim and Swati also set up a plate to test our arsenic sensing parts at different concentrations of arsenic – they ran a plate with a blank LB column, five control columns (JG700, MR-1, p39k, p40k, and p41k), and three columns of each of our arsenic-sensitive strains. To each row they added a different concentration of arsenic, going from 0uM to 5uM arsenic. The final OD of 100uL in each well was 0.8, and the plate was left overnight in the plate reader. | ||
+ | <br><br> | ||
+ | <b>Conjugating nah into Shewy:</b> | ||
+ | Caleb miniprepped the 36 hour 30 mL culture of colony N. The size of the pellet after the first spin step was similar to the size of a ~5mL normal culture, consequently, the yield was only 96.7 ng/uL. | ||
+ | <br><br> | ||
+ | <b>September 27th</b> | ||
+ | <br><br> | ||
+ | <b>Running Reactors:</b> For the first time, we added arsenic to our reactors. Specifically, once Dylan saw that the two reactors inoculated with an arsenic reporter strain (JG700+p14k) had been producing steady current for several retention times, he dosed the media vessel to a final concentration of 10 μM sodium arsenite. | ||
+ | <br><br> | ||
+ | <b>Conjugating nah into Shewy:</b> | ||
+ | Fortunately, there was enough DNA for Caleb to submit the miniprepped DNA from colony N for Sanger sequencing today. Tina started 3 x (1mL cultures of colony N). | ||
+ | <br><br> | ||
+ | <b>Fluorescence tests:</b> Claire and Swati set up another control plate with constitutively produced mRFP in both WM3064 and JG700. Since using 100uL starting at and OD of 0.8 seemed to work well for the arsenic plate, we set up the control plate with the same parameters and left it in the plate reader overnight. | ||
+ | <br><br> | ||
+ | <b>September 28th</b> | ||
+ | <br><br> | ||
+ | <b>Running Reactors:</b> Since several retention times passed without any current response to 10 μM sodium arsenite, Dylan increased the concentration in the media vessel, hitting our reactors with 100 μM arsenite. | ||
+ | <br><br> | ||
+ | <b>Fluorescence tests:</b> We noticed that the arsenic data did not seem to have any signal above background, and realized that it may have been because the plate reader wasn’t set to read a clear-bottomed plate. We concluded that, since from the test done on the 27th we could see distinct difference between fluorescence levels of constitutively produced mRFP, our experimental procedure is good and the plate reader was just on the wrong setting. Additionally, since the bioreactors are not getting upregulation of arsenic up to 10uM, we decided next time to test higher concentrations of arsenic (0uM to 500uM) | ||
</div> | </div> | ||
</div> | </div> |
Revision as of 03:18, 4 October 2012
Wet Lab - September
-
Week 1
Conjugating nah into Shewy: The mysterious failure of past week's ligation lead to a redigestion (in case we run out of DNA) and religation of last week's digestions. Nah to oriT was ligated in ratios of 3:1 and 6:1 - when run on a gel, bands once more failed to appear... But wait! Epiphany! The team realized that we forgot to dephosphorylate the oriT backbone! After that silly mistake, 1:1, 3:1, and 6:1 ratios of nah:oriT were set up in a ligation mixture and electroporated into WM3064. Once again, slow growth and tiny colonies were observed - perhaps due to the strain of putting such a massive operon in a cell. 13 colonies were deemed large enough to try colony PCR, and 3 of them definitely showed bands that corresponded to the length of our construct. We set up liquid cultures for Miniprepping and once again observed slow growth. We tried subculturing - but still, general opaqueness. Miniprepping proceeded anyway, as we are a fairly optimistic bunch (and the cultures were by then over 24 hours old).
Site-directed mutagenesis: After several more unsuccessful attempts, lengthy discussion among teammates, and consultation with several graduate advisors, the team decided to put this subproject on hold for the time being. Swati finished up with one last attempt, then joined Claire for fluorescence testing. That's all, folks! Daily DetailsWeek 2
Conjugating nah into Shewy: Unfortunately, the miniprep yields from last week were all low except one shining colony - which didn't have the nah operon in it. We ran it to test our hypothesis that slow growth of E Coli was due to the added stress on the cell of having such a large operon in it - our data so far matches our hypothesis, as that colony grew faster in culture and the plate and also yielded substantially more DNA after miniprepping.
Site-directed mutagenesis: Though we were unable to confirm the presence of the nah operon with sequencing (as the miniprep failed), we decided to proceed with conjugation into Shewanella just in case the transformation succeeded. We tried to grow one of the colonies with a very strong band from last week's gel in a liquid culture, and once again, observed slow growth. We conjugated into JG700 and JG700 with SAL, a plasmid with our salicylate reporter system. Liquid cultures were made from the conjugation - unfortunately, there was no growth. We streaked new plates with the old transformation in the hopes that something would finally grow! Daily DetailsWeek 3
Conjugating nah into Shewy: Some colonies were observed from last week's restreaking! A massive 15 colony liquid culture was set up, but to no avail. After five long days, we were forced to conclude that conjugation failed. Sadface. Good thing we had extra 3:1 nah to oriT ligation sitting around, which we transformed into both WM3064 and DH5a, the latter of which we hoped would grow faster. 16 colonies from the Sep 4th ligation were also restreaked. And lo! Every single restreak had colonies the next day, which warranted a massive 16 colony colony PCR (try saying that five times fast). Interestingly, the WM3064 plates grew, but the DH5a transformation failed, which we concluded was due to a bad electrocompetent cell stock (we noticed weird white precipitate in the cell mixture before). Daily DetailsWeek 4
Conjugating nah into Shewy: A gel of last week's colony PCRs hinted that one of the colonies had the nah operon in it! Excitement! Liquid cultures were made, and there was just enough DNA after miniprepping to submit for sequencing. We will collectively hold our breaths and wait for positive results.
This week, we were able to begin characterizing the current response to arsenic of S20 (JG700 + p14k; see strain list). Additionally, we submitted physical DNA for six BioBrick parts to the parts registry. Daily Details -
Week 1
Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum.Week 2
Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum.Week 3
Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum.Week 4
Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum. -
Week 1
It has been said that astronomy is a humbling and character-building experience. There is perhaps no better demonstration of the folly of human conceits than this distant image of our tiny world. To me, it underscores our responsibility to deal more kindly with one another, and to preserve and cherish the pale blue dot, the only home we've ever known.Daily Details:
Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum.Week 2
It has been said that astronomy is a humbling and character-building experience. There is perhaps no better demonstration of the folly of human conceits than this distant image of our tiny world. To me, it underscores our responsibility to deal more kindly with one another, and to preserve and cherish the pale blue dot, the only home we've ever known.Daily Details:
Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum.Week 3
It has been said that astronomy is a humbling and character-building experience. There is perhaps no better demonstration of the folly of human conceits than this distant image of our tiny world. To me, it underscores our responsibility to deal more kindly with one another, and to preserve and cherish the pale blue dot, the only home we've ever known.Daily Details:
Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum.Week 4
It has been said that astronomy is a humbling and character-building experience. There is perhaps no better demonstration of the folly of human conceits than this distant image of our tiny world. To me, it underscores our responsibility to deal more kindly with one another, and to preserve and cherish the pale blue dot, the only home we've ever known.Daily Details:
Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum.