Team:Caltech/week5

From 2014.igem.org

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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>
         <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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     <li>Began assembly of agr reception system:
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     <li>Began assembly of Agr reception system:
     <ul><li>PCRed backbones for pAA008, pAA009, and pAA010 plasmids to include overlaps to be used in Gibson.
     <ul><li>PCRed backbones for pAA008, pAA009, and pAA010 plasmids to include overlaps to be used in Gibson.
         <ol style="list-style-type:lower-roman"><li>pAA008: insert contains agrC (receptor) with 4 SH3 domains (part of scaffold system)</li>
         <ol style="list-style-type:lower-roman"><li>pAA008: insert contains agrC (receptor) with 4 SH3 domains (part of scaffold system)</li>
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<b>Export Systems</b>
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<ul><li>Colonies were picked from the 4 plates that grew colonies of the 5 incubated overnight (attempting to clone plasmids pTG002-pTG006) and resuspended in 10 &mu;L water.</li>
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    <li>Colony PCR was run on the colony resuspensions.
 +
        <ul><li>Results appear to suggest transformation of: 1 colony with pTG003 (agrBD with His-tag on C-terminus of ligand agrD), 2 colonies with pTG004 (lamBD), 4 colonies with pTG005 (fsrB with His-tag on N-terminus of ligand), and 1 colony with pTG006 (fsrB with His-tag on C-terminus of ligand)</li></ul>
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    <li>Liquid cultures were prepared for the most promising colonies as screened by colony PCR and incubated overnight</li>
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<b>agrBCDA Reception System and Combinatorial Promoters</b>
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<ul><li>Reattempt to clone pAA009 & pAA010 for the Agr system.
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    <ul><li>Redid PCR of pAA009/pAA010 backbone. PCR product was then DpnI digested and PCR-purified.</li>
 +
        <li>Gibson assembly of pAA009 and pAA010 using the PCRed backbone and the respective AgrA geneblock</li>
 +
    </ul></li>
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    <li>Combinatorial Promoters:
 +
    <ul><li>Colony PCR run on 6 colonies transformed with pAA003.</li>
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        <li>Liquid cultures were prepared for those colonies that were deemed promising based on colony PCR screening.</li>
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        <li>Additionally, jwAA001 liquid cultures with inducers (aTc & IPTG) had been allowed to continue growing last night. Fluorescence measurements were repeated this morning and appeared to confirm the AND-gate logic we had desired</li>
 +
        <li>New overnight liquid culture of jwAA001 was set up for experimentation with more inducer concentrations later this week</li>
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    </ul></li>
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</ul>
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Revision as of 23:09, 17 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:
    • PCRed backbones for pAA008, pAA009, and pAA010 plasmids to include overlaps to be used in Gibson.
      1. pAA008: insert contains agrC (receptor) with 4 SH3 domains (part of scaffold system)
      2. pAA009: insert contains 2 components: GFP regulated by a P2 promoter and agrA (response regulator) linked to an SH3 peptide (other part of scaffold system).
      3. pAA010: identical to pAA009 but does not contain SH3 peptide/scaffold at end of agrA
    • DpnI digested PCRed backbones and ran a gel to confirm presence of backbone. pAA008 backbone appears to have been successfully constructed, but the backbone for pAA009/pAA010 apparently failed (need to redo).
    • Gibson assembly of pAA008 backbone with geneblock containing modified agrC sequence

Wednesday, 7/16/14

Export Systems
  • Colonies were picked from the 4 plates that grew colonies of the 5 incubated overnight (attempting to clone plasmids pTG002-pTG006) and resuspended in 10 μL water.
  • Colony PCR was run on the colony resuspensions.
    • Results appear to suggest transformation of: 1 colony with pTG003 (agrBD with His-tag on C-terminus of ligand agrD), 2 colonies with pTG004 (lamBD), 4 colonies with pTG005 (fsrB with His-tag on N-terminus of ligand), and 1 colony with pTG006 (fsrB with His-tag on C-terminus of ligand)
  • Liquid cultures were prepared for the most promising colonies as screened by colony PCR and incubated overnight
agrBCDA Reception System and Combinatorial Promoters
  • Reattempt to clone pAA009 & pAA010 for the Agr system.
    • Redid PCR of pAA009/pAA010 backbone. PCR product was then DpnI digested and PCR-purified.
    • Gibson assembly of pAA009 and pAA010 using the PCRed backbone and the respective AgrA geneblock
  • Combinatorial Promoters:
    • Colony PCR run on 6 colonies transformed with pAA003.
    • Liquid cultures were prepared for those colonies that were deemed promising based on colony PCR screening.
    • Additionally, jwAA001 liquid cultures with inducers (aTc & IPTG) had been allowed to continue growing last night. Fluorescence measurements were repeated this morning and appeared to confirm the AND-gate logic we had desired
    • New overnight liquid culture of jwAA001 was set up for experimentation with more inducer concentrations later this week

Thursday, 7/17/14

Friday, 7/18/14