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console.js:26

Week2: May 27 Out of Lab

Friday, 5/27

LynnMembers present: Celine (for the first half hour), Tam (left at 3:30), Hamed, Cathy, DK, Alex, Katariina

Lab Stuff: (research, plasmid design, protocol design, troubleshooting)

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Research into the appropriate promoter:

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"Transcription of gesABC is delayed compared with the Au-induced expression of golTS or golB, and requires higher GolS intracellular levels, suggesting a hierarchical activation of the gol regulon to cope with contaminated environments."

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In the presence of gold ions: There is a sensitivity/specificity trade-off between using pgolB and pges (http://www.ncbi.nlm.nih.gov/pubmed/17919284: see below)

	A	B	C
1		pgolB	pges
2	Sensitivity (i.e. Transcription level in presence of gold(III) ions)	Higher (36 folds) i.e. easier to set off	Lower i.e. harder to set off
3	Specificity (i.e. Response to Cu(II))	Lower i.e. more sensitive to Cu(II)	Higher (65 folds) i.e. less sensitive to Cu(II)

Table1

Sensitivity:

clipboard_2016-05-27_11:31:47.png

Time-course of GolS induction of gesABC and golB transcription in the presence of Au(III) (AuHCl₄). The maximum amount of transcription response shows 278 Miller units for gesABC and 10090 Miller units for golB - 36-fold difference. There is an indication that pGesABC was less sensitive to pGolB in the presence of Au(III) as pGesABC transcription level was significantly low in the first 5 hours. Transcription reaches the maximum capacity at 5.8 hours for gesABC and 3.6 hours for GolB.

Specificity:

clipboard_2016-05-27_11:15:44.png

GesABC::LacZ fused gene expresses no response to the addition of CuSO₄ (Cu(II)) in the system. GolB::LacZ fused gene expresses a 65-fold response to the addition of CuSO₄ (Cu(II)) in the system.

In general, there is a high level of sensitivity to Au ions in pGolB than in pGesABC. However, pGesABC shows a higher specificity to Au ions than Cu ions in comparision to pGolB which can be induced by both Au and Cu ions.

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Creation of golS variants in the feature library using the following table to assist in optimizing the new codons:

E. coli Codon Bias.gif

Administrative: (shipment orders, inventory)

Email Updates: (Correspondance with proffessors, shipping companies, etc)

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Followed up regarding lockers

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Drafted e-mail to Kayla and sent to Umar for review before sending it out

Tasks to Follow up on - leading into week 3

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Promoter debacle (specificity/sensitivity) - All

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Luciferase/RFP (reaction with cellulose) - Kat

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merR family and Nickle sensors generally (as back up) - Wiki team

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Outlining Dr. Pardee's updated protocol - Kat

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golS structures (4 versions) - Tam

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118 - Kat

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113 - Alex

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113 + 118 - Alex

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CupR - Cathy

Tasks leading into week 3 - v2.jpg

Meeting Notes: