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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 μM 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 μM IPTG, 100 ng/mL aTc. </li> |
| </ul></li> | | </ul></li> |
- | <li>Began assembly of agr reception system: | + | <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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| </td> | | </td> |
| <td valign="top"> | | <td valign="top"> |
- | <br> | + | <b>Export Systems</b> |
| + | <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 μL water.</li> |
| + | <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> |
| + | <li>Liquid cultures were prepared for the most promising colonies as screened by colony PCR and incubated overnight</li> |
| + | </ul> |
| + | <b>agrBCDA Reception System and Combinatorial Promoters</b> |
| + | <ul><li>Reattempt to clone pAA009 & pAA010 for the Agr system. |
| + | <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> |
| + | <li>Combinatorial Promoters: |
| + | <ul><li>Colony PCR run on 6 colonies transformed with pAA003.</li> |
| + | <li>Liquid cultures were prepared for those colonies that were deemed promising based on colony PCR screening.</li> |
| + | <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> |
| + | </ul></li> |
| + | </ul> |
| </td> | | </td> |
| </tr> | | </tr> |
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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
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Week Five |
Monday, 7/14/14
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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.
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Tuesday, 7/15/14
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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.
- pAA008: insert contains agrC (receptor) with 4 SH3 domains (part of scaffold system)
- 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).
- 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
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Wednesday, 7/16/14
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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
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Thursday, 7/17/14
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Friday, 7/18/14
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