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From 2010.igem.org

Week5 Highlights

Our LacP-GFP construct expressed fluorescence. We received a sponsorship from NEB. We also me with Wei Zhang and decided to grow our biofilm on normal agar plates rather than in a flow cell. Our first 3A assembly was successful, but all of our following 3A assemblies failed. We began making competent TOP10 cells. CHS3 was successfully PCRed and purified.

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7/11/10

Roughly 60% of 1-12O colonies expressed green fluorescence Only a few colonies of 1-12M expressed green fluorescence No red fluorescence was seen.

Miniprepped DNA (LacP1-GFP construct) --> stored in -20°C

IPTG seems unnecessary for expression of GFP via the LacP promoter - very leaky --> need LacL repressor

Ran PCR products in gel -> good yield

Kit to Stock:

• 1-18I (e0430: YFP)
• 1-18F (e1010: RFP)
• 1-20L (q001121: LacPI1)
• 1-20P (q04121: LacPI2)
• 1-10H (i712019: Luciferase)
• 1-3A (psb1c3: BBPlasmid-C)
• 1-5A (psb1k3: bbplasmid-K)
• 1-7A (psb1T3: bbplasmid-T)
• 1-1C (psb1A3 bbplasmid-A)

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7/12/10

All backbone plasmid transformation colonies appear reddish/purple (due to RFP gene in plasmids)

Colonies were selected from the yfp (e0430 1-18I), rfp (E1010 1-18F), lacpl1 (Q001121 1-20L), lacpl2 (Q04121 1-20P), luciferase (I712019 1-10H), bbplasmid-c (psb1c3 1-3A), bbplasmid-k (psb1k3 1-5A), bbplasmid-t (psb1T3 1-7A), bbplasmid-A (psb1A3 1-1C), 1-2I, and 2-6K plates and incubated in LB broth at 37C for 16 hours (starting at 6:00pm)

Received sponsorship from NEB.

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7/13/10

Miniprep --> mainly ~200ng/ul (3 parts that we redid were still low = ~50ng/ul)

Used 3A assembly to assemble LacP1-GFP, LacP2-GFP, and LacPI1-GFP (in 4 different backbone plasmids, psb1A3/psb1T3/psb1C3/psb1K3, made from transforming from well parts)

Met with Wei Zhang to discuss biofilm growth. Decided flow cells would not be the most cost effective and useful way to grow our E.Coli biofilm. Plan on using normal agar plates. Discovered that BacLight Live/Dead assay can be used on bacteria grown on a plate rather than bacteria growth in broth. We were told to add the BacLight dye to the lawn of bacteria and allow for it to diffuse before washing with ______.

Received sponsorship from Promega.

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7/14/10

None of the assemblies grew.

Group Meeting:

• Need to keep notes for future team (i.e. sponsorships, changes to protocol, etc.)
• Made a list of things to get from Promega

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7/15/10

PCR Purified CHS3 PCR product

Ran 3 PCR reactions using Amp, Chl, Tet linearized backbone plasmids as the template

3A Assembly of CP1-GFP, LacP1-GFP, LacP2-GFP, LacPI1-GFP, LacPI2-GFP, and CHS3-XX.

Streaked TOP10 cells on agar plates and incubate overnight for making competent cells.

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7/16/10

None of the assemblies grew.

PCR Purified backbone plasmid PCR products.

3A Assembly of CP1-GFP, LacP-GFP, LacPI1-GFP.

Selected single TOP10 colonies and prepared seed stocks for making competent TOP10 cells.

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