Team:Northwestern/Notebook/Week5

From 2011.igem.org

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<DIV style="font-size:20px">
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Day 20 - Monday, July 11th 2011
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</DIV>
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----------------------
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*The transformations from the weekend had some very weird results. They were all red despite our attempts to ligate GFP. We think the issues is with our Kan backbones (uncut backbones may have transformed the bacteria)
 +
*We began another batch of competent cells, using a slightly different procedure from a molecular cloning manual.
 +
*We miniprepped the overnight cultures containing the RBS, the Tet and Chlor backbones, and two Rhl related parts
 +
*We finished designing primers to extract Las/Rhl promoters from the genome
 +
*We began a PCR extraction of the Las and Rhl genes from the genome.
 +
*Ran the PCR products out on a gel to see if the process was successful
 +
<!--and then two lines between this entry and the next entry-->
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<DIV style="font-size:20px">
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Day 21 - Tuesday, July 12th 2011
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</DIV>
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----------------------
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*Our SOB broth was contaminated! The test tube we grew overnight containing just broth was still cloudly in the morning. As a result, our competent cell procedure will have to be restarted and new SOB will have to be made.
 +
*Because the overnights had an antibiotic, we decided to miniprep anyway despite the contamination.
 +
*We started a digest of our RBS parts and our eventual downstream parts so that we can ligate them together via a modified 1A assembly. The procedure leaves the promoter in with its backbone, making gel extraction possible.
 +
*We also again attempted to digest and ligate our promoters together with our reporters. This will allow us to test if our promoters are working.
 +
*We attempted PCR again to extract RhlR.
 +
 +
 +
<DIV style="font-size:20px">
 +
Day 22 - Wednesday, July 13th 2011
 +
</DIV>
 +
----------------------
 +
*Ran the PCR products from yesterday on a gel. This time, neither gene appeared to be successfully amplified. All we saw were two small, unidentified bands.
 +
*Competed cells procedure restarted with fresh, uncontaminated SOB
 +
*We transformed yesterday’s ligations.
 +
*We started a new 3A assembly of our promoters, receptors and reporters. We tried using double the suggested amount of DNA (1ug) to improve the yield. These parts were ligated and transformed in the afternoon.
 +
*We also started a modified 3A Assembly with similar parts. This procedure requires gel extraction, which took longer, so the ligation was left to run overnight.
 +
*We ordered the sequencing primers and the primers that will allow us to extract the promoters from the genome.
 +
 +
 +
<DIV style="font-size:20px">
 +
Day 23 - Thursday, July 14th 2011
 +
</DIV>
 +
----------------------
 +
*Miniprepped 28 overnights of all of our parts and backbones. The DNA concentrations were generally very good, among the best that we’ve had so far.
 +
*Many of our ligated transformations grew! There were colonies on almost every plate. This is a good sign, although we will have to sequence and/or run a gel to make sure the cells were transformed with the correct plasmid.
 +
*We also transformed the rest of our ligations that had ligated overnight
 +
*We finished making the new competent cells, aliquoted them, and did a test transformation with a positive control.
 +
*Made new Tet and Amp plates
 +
*Decided to start overnights from the successful transformations, but only for the parts with Ribosome Binding Sites. Our advisors definitely think this will improve efficiency.
 +
 +
 +
<DIV style="font-size:20px">
 +
Day 24 - Friday, July 15th 2011
 +
</DIV>
 +
----------------------
 +
*Miniprepped the overnights of our ligated RBS parts.
 +
*Started a PCR reaction with our new primers to hopefully extract Las and Rhl sensitive promoters directly from the pseudomonas genome. We also attempted to extract RhlR again.
 +
*Cut our ligated and miniprepped overnights with different restriction enzymes and ran it on a gel to confirm the presence of two P restriction sites.

Revision as of 15:36, 9 August 2011

RETURN TO IGEM 2010


Day 20 - Monday, July 11th 2011

  • The transformations from the weekend had some very weird results. They were all red despite our attempts to ligate GFP. We think the issues is with our Kan backbones (uncut backbones may have transformed the bacteria)
  • We began another batch of competent cells, using a slightly different procedure from a molecular cloning manual.
  • We miniprepped the overnight cultures containing the RBS, the Tet and Chlor backbones, and two Rhl related parts
  • We finished designing primers to extract Las/Rhl promoters from the genome
  • We began a PCR extraction of the Las and Rhl genes from the genome.
  • Ran the PCR products out on a gel to see if the process was successful


Day 21 - Tuesday, July 12th 2011

  • Our SOB broth was contaminated! The test tube we grew overnight containing just broth was still cloudly in the morning. As a result, our competent cell procedure will have to be restarted and new SOB will have to be made.
  • Because the overnights had an antibiotic, we decided to miniprep anyway despite the contamination.
  • We started a digest of our RBS parts and our eventual downstream parts so that we can ligate them together via a modified 1A assembly. The procedure leaves the promoter in with its backbone, making gel extraction possible.
  • We also again attempted to digest and ligate our promoters together with our reporters. This will allow us to test if our promoters are working.
  • We attempted PCR again to extract RhlR.


Day 22 - Wednesday, July 13th 2011

  • Ran the PCR products from yesterday on a gel. This time, neither gene appeared to be successfully amplified. All we saw were two small, unidentified bands.
  • Competed cells procedure restarted with fresh, uncontaminated SOB
  • We transformed yesterday’s ligations.
  • We started a new 3A assembly of our promoters, receptors and reporters. We tried using double the suggested amount of DNA (1ug) to improve the yield. These parts were ligated and transformed in the afternoon.
  • We also started a modified 3A Assembly with similar parts. This procedure requires gel extraction, which took longer, so the ligation was left to run overnight.
  • We ordered the sequencing primers and the primers that will allow us to extract the promoters from the genome.


Day 23 - Thursday, July 14th 2011

  • Miniprepped 28 overnights of all of our parts and backbones. The DNA concentrations were generally very good, among the best that we’ve had so far.
  • Many of our ligated transformations grew! There were colonies on almost every plate. This is a good sign, although we will have to sequence and/or run a gel to make sure the cells were transformed with the correct plasmid.
  • We also transformed the rest of our ligations that had ligated overnight
  • We finished making the new competent cells, aliquoted them, and did a test transformation with a positive control.
  • Made new Tet and Amp plates
  • Decided to start overnights from the successful transformations, but only for the parts with Ribosome Binding Sites. Our advisors definitely think this will improve efficiency.


Day 24 - Friday, July 15th 2011

  • Miniprepped the overnights of our ligated RBS parts.
  • Started a PCR reaction with our new primers to hopefully extract Las and Rhl sensitive promoters directly from the pseudomonas genome. We also attempted to extract RhlR again.
  • Cut our ligated and miniprepped overnights with different restriction enzymes and ran it on a gel to confirm the presence of two P restriction sites.
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