Team:Caltech/week7

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Notebook
Overview

Week 1

Week 2

Week 3

Week 4

Week 5

Week 6

Week 7

Week 8

Week 9

Week 10

Week 11

Week 12

Week Seven

Monday, 7/28/14

Export Systems
  • Ran colony PCR on pTG002/3/6-transformed JM109 cultures grown up over the weekend. Bad results for all of them
  • Created and ordered new primers that will hopefully avoid secondary-structure-formation when the exonuclease attacks the 5' end during Gibson assembly.
lamBCDA & fsrABC Reception Systems
  • Single-transformations run on each of the constructs used in the double-transformations:
    1. pMB001
    2. pMB002
    3. pMB003
    4. pMB004
    5. pMB005
    6. pMB006
agrBCDA Reception System and Combinatorial Promoters
  • Colonies were picked from pAA008 transformations incubated over the weekend, as well as the pAA009 & pAA010 transformations, which had been set up last Thursday.
  • Set up liquid cultures of the colonies to grow overnight in preparation for miniprep and sequencing tomorrow

Tuesday, 7/29/14

Export Systems
  • Ran Gibson assembly products on a gel. Results inconclusive
  • Started 3 mL liquid cultures of bacteria (with pTG004 & pTG005) with varying levels of aTc induction:
    • 0 nM, 50 nM, 150 nM, 250 nM, 350 nM, & 450 nM
lamBCDA & fsrABC Reception Systems
  • Colony PCR was run on several of the colonies that emerged from the single-transformations last night
  • Gel was run, but wrong primers were used. New primers for sequencing/colPCR were ordered, and colPCR will be redone tomorrow.
agrBCDA Reception System and Combinatorial Promoters
  • Miniprepped liquid cultures grown last night and shipped samples for sequencing.

Wednesday, 7/30/14

Export Systems
  • Ran new PCR reactions with newly designed primers to extract backbones of pTG002/3/6.
  • DpnI-digested (4 hrs) and PCR-purified the PCR products.
  • Products were run on a gel to confirm their creation.
  • Gibson assembled the backbones with their geneblock inserts
  • Gibson assemblies were transformed into JM109, which were plated & incubated overnight.
Additionally we began experimentation on the pTG004 (lam system) & pTG005 (fsr system) constructs we had successfully constructed in Week 5, using the liquid cultures induced with aTc yesterday.
  • Western blots were run on both supernatant and cell lysate samples (6 concentrations of aTc, so 12 Western lanes for each system) from the 2 liquid cultures grown up last night. 3xFLAG-antibodies appear to mark proteins in the fsr supernatant and cell lysate lanes.
lamBCDA & fsrABC Reception Systems
agrBCDA Reception System and Combinatorial Promoters
  • Analyzed sequencing results:
    • pAA008 successfully constructed
    • Minipreps supposedly containing pAA009 & pAA010 instead contain pKS001 backbone template
  • We redid PCR of the pAA009/010 backbone
  • DpnI digestion for 2 hours, followed by PCR purification. The purified product was then run on a gel.

Thursday, 7/31/14

Export Systems
lamBCDA & fsrABC Reception Systems
agrBCDA Reception System and Combinatorial Promoters
agrBCDA system:
  • We used a gel extraction kit to extract the pAA009/010 backbone from the gel run yesterday.
Combinatorial Promoters:
  • PCRed the plasmid backbone and combinatorial promoter parts of pAA002.
  • DpnI digested the PCR products
  • Ran a gel on the products to confirm sequences were of the correct length
  • PCR-purified the DpnI-digested products

Friday, 8/1/14

Export Systems
lamBCDA & fsrABC Reception Systems
agrBCDA Reception System and Combinatorial Promoters
  • Did a Gibson assembly of pAA002 (containing a combinatorial promoter), pAA009, and pAA010 (containing agr reception system), using PCRed/gel-extracted (respectively) products from yesterday.