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 13

Week 14

Week 15

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.
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
  • 5 colonies from each of the overnight transformations were picked (15 total), and we ran colony PCR (52°C annealing step) on them using the newly ordered sequencing primers.
  • colony PCR products were run on a gel, showing that none of them contained the correct construct.
  • 10 new colonies were picked and "colony-PCR-ed" (this time with a 47° annealing step). However, running the product on a gel showed these didn't work either.
lamBCDA & fsrABC Reception Systems
  • colony PCR was redone on the pMB001-006 transformations using the newly ordered primers. Still no binding.
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
  • To debug our colony PCRs from yesterday, we set up 8 colony-PCR reactions on a annealing temperature gradient, using E. coli containing pKS001 (the positive control in our earlier colPCRs) as the template.
    • Annealing temperature was allowed to range from 47°C to 54°C
    • Optimal annealing temperature for our new sequencing primers was determined to be around 48.6°C, with no binding beyond 52°C.
  • To create a negative control for our Western blots, we obtained a pTet_GFP plasmid that had been assembled via Golden Braid assembly. This assembly product was transformed into JM109 and plated on a chloramphenicol plate.
    • We will eventually place a 3xFLAG behind the GFP in this plasmid to be used in a Western.
  • In preparation for the LCMS run to detect the lam system's signalling peptide, we are going to remove the 3xFLAG from pTG004 (as it would be in any actual signal-reception system involving lam we would design):
    • 3xFLAG was PCRed out (linearizing the pTG004 plasmid) using 2 new primers we had designed with phosphorylated 5' ends. 50°C annealing step was used.
    • PCR-purified PCR product.
    • Around-the-horn ligation (to recircularize pTG004-without-FLAG) was run using Quick Ligation kit
    • Ligation product was transformed into JM109, plated on carbenicillin, and incubated.
    • Colony PCR (49°C annealing step) was then run on the 3 colonies that grew up.
    • A gel was run on the colony PCR products, revealing ____
lamBCDA & fsrABC Reception Systems
  • Still no success with the colony PCR.
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.