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From 2010.igem.org

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Contents 1 8/9/10 2 8/10/10 3 8/11/10 4 8/12/10 5 8/13/10

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 in ________

Grew bakers yeast to test for presence of chitin

Colony PCR on (pMal-CHS3)

CP-LacPI part testing

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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

Digested __________ (sean)

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 --> should see 1 2500kb band

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

Cycles 1 of File:MAGE for tqsA Knockouts.

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