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From 2010.igem.org

Week9 Highlights

We bought chitosan powder form a local health store to use as a positive control for chitin staining. We ran a colony PCR on pMAL-CHS3 and found no positive results. We began MAGE knockouts of tqsA. Discovered that our Holin1, Holin2, pMAL-CHS3, and CP-LacPi parts were faulty. We also decided to use a rhodamine-conjugated chitin probe as an alternative chitin stain because calcofluor white resulted in excess background.

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8/9/10

Bought Chitosan powder to use as a positive control for calcofluor white --> glowed bright blue/green when stained

Stained (pMal-CHS3) ligation products for the presence of chitin --> ligation 1/tube 3 had slight fluorescence

Used UV-vis Spectroscopy to measure chitin concentrations

Grew bakers yeast to test for presence of chitin

Colony PCR on (pMal-CHS3)

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8/10/10

Ran gel for colony PCR no positives

Ran PCR for CHS3 insert into pMal vector

Stained varying concentrations of chitosan (.25g/ml, 25ug/ml, 5ug/ml, 1ug/ml) and (pMal-CHS3) cells --> cells showed no signs of chitin

Ligated (CP-LacPi)-(GFP) in Tet backbone

Made LB minimum media for electroporated cells

Miniprepped circular backbone plasmid

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8/11/10

Digested and ran a gel of CHS3 and backbone plasmids (C,T,K,A)

Tested restriction enzymes by digesting circular plasmids with each enzyme individually --> saw 1 2500kb band which was expected

Organized the DNA samples in our -20.

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8/12/10

Weekly meeting

• Went over our gels --> identified faulty parts (holin1, holin2, CHS3-pMal, CP-LacPI)
• Plan to re-kit to stock and reassemble

Decided to use an alternative stain because calcofluor results in too much background and our confocal microscope can not excite in the UV spectrum

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8/13/10

Methanol fixation of yeast and E.Coli cells --> stained with rhodamine-conjugated chitin probe (allowed to incubate overnight)

Competency of ChiA knockouts = 2.5x10^7 and 5.0x10^6

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