Team:Caltech/week5

From 2014.igem.org

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<ul><li>Grew liquid cultures of <i>E. coli</i> transformed with pWW2149+pWW1521 and pWW2149+pWW1523 (colonies had been allowed to grow on agar plates over the weekend)</li>
<ul><li>Grew liquid cultures of <i>E. coli</i> transformed with pWW2149+pWW1521 and pWW2149+pWW1523 (colonies had been allowed to grow on agar plates over the weekend)</li>
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<b>agrBCDA Reception System and Combinatorial Promoters</b>
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<b>Combinatorial Promoters</b>
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<ul><li></li>
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<ul><li>Set up overnight liquid cultures of DH5&alpha;-Z1 transformed with pAA001 (with pTet-pLac combinatorial promoter)</li>
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    <li>Transformed DH5&alpha;-Z1 with Gibson product (aimed at assembling pAA003--containing pBAD-pTet combinatorial promoter) created Friday.</li>
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</ul>
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<b>agrBCDA Reception System and Combinatorial Promoters</b>
<b>agrBCDA Reception System and Combinatorial Promoters</b>
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<ul>
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<ul><li>Began experiments on bacteria transformed with pAA001, testing different inducer concentrations:
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    <ul><li>([IPTG] in &mu;M, [aTc] in ng/mL): (0,0); (0,100); (100,0); (100,100)</li>
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        <li> Preliminary results promising: GFP <a href="2014.igem.org/Team:Caltech/Results">significantly expressed</a> [include link to the graph] only at 100 &mu;M IPTG, 100 ng/mL aTc. </li>
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    </ul></li>
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    <li>Began assembly of agr reception system:
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    <ul><li>
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Revision as of 18:34, 16 July 2014


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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 Five

Monday, 7/14/14

Export Systems
  • Redid Gibson assembly of comX constructs to reattempt cloning
  • Constructs transformed into JM109, plated on carbenicillin, and incubated overnight
lamBCDA & fsrABC Reception Systems
  • Grew liquid cultures of E. coli transformed with pWW2149+pWW1521 and pWW2149+pWW1523 (colonies had been allowed to grow on agar plates over the weekend)
Combinatorial Promoters
  • Set up overnight liquid cultures of DH5α-Z1 transformed with pAA001 (with pTet-pLac combinatorial promoter)
  • Transformed DH5α-Z1 with Gibson product (aimed at assembling pAA003--containing pBAD-pTet combinatorial promoter) created Friday.

Tuesday, 7/15/14

Export Systems
  • ComX System
    • Ran colony PCR again on overnight colonies with reassembled comX constructs (3 colonies picked from each of the 5 plates)
    • Ran a gel on the PCR products. Resulting gel.
  • agrBD, lamBD, & FsrB Systems
    • PCR assembly of backbones with overhangs needed for assembly of agrBD, lamBD, & FsrB export systems
    • DpnI digestion of PCR product for 2 hours and then PCR purification were run to remove any non-backbone fragments of DNA
    • The five export constructs to test [link to images of the 5 plasmids we constructed] were assembled via Gibson using the PCRed backbones and geneblocks (containing the inserts) that arrived yesterday afternoon
    • 1 μL of each of the 5 Gibson products was then transformed into JM109. The bacteria were then plated on 5 carbenicillin plates & incubated @ 37°C overnight
lamBCDA & fsrABC Reception Systems
  • Testing the Scaffold System
    • Created glycerol stocks of the overnight liquid cultures (with bacteria transformed with pWW2149+pWW1521 and pWW2149+pWW1523) and stored them at -80°C
    • The remainder of the overnight cultures were then grown in clear MOPS media
    • Different concentrations of arabinose [specifically what concentrations?] were added to separate aliquots of the cultures in a 96-well plate
    • GFP fluorescence data was collected from these colonies in a plate reader over [how many hours post-induction?]
  • Construction of lam & fsr Reception Systems
    • PCRed pKS001 template to extract vector backbone with overhangs for later Gibson assembly
agrBCDA Reception System and Combinatorial Promoters
  • Began experiments on bacteria transformed with pAA001, testing different inducer concentrations:
    • ([IPTG] in μM, [aTc] in ng/mL): (0,0); (0,100); (100,0); (100,100)
    • Preliminary results promising: GFP significantly expressed [include link to the graph] only at 100 μM IPTG, 100 ng/mL aTc.
  • Began assembly of agr reception system:

Wednesday, 7/16/14


Thursday, 7/17/14

Friday, 7/18/14