iGEM%20Bioware

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iGEM%20Bioware

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iGEM Bioware Week 9 Arsenic Bioremediation Got ArsR into plasmid backbone and began site directed mutagenesis. using NEB competent cells significantly improved ... – PowerPoint PPT presentation

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Title: iGEM%20Bioware

1
iGEM Bioware

Week 9

2
Arsenic Bioremediation

Got ArsR into plasmid backbone and began site
directed mutagenesis.
using NEB competent cells significantly improved
efficiency.
PCR of ArsB and LamB is still not working--Any
suggestions? Both these proteins are vital to
our project.
Ordered new primers for RpoS promoter and ArsR
Promoter with EcoRI cut site to increase yield
and efficiency.
Also worked to get constitutive promoters in
front of gas vesicles biobrick but getting lawns
after transformations.

3
Expected SizespRpoS438 438 bppRpoS232 232
bpArsB 1290 bpLamBF1R 1.3 kbLamBF2R 1.3
kbArsR fusion 350 bpArsR 354 bppArsRF1R1
540 bppArsRF1R2 547 bppArsRF2R1 293
bppArsRF2R2 300 bp
ArsB
LamB F2R

pRpoS
438

ArsR
pArsR F1R1
pArsR F1R2
pRpoS 232
LamB F1R
ArsR Fusion
500 bp
pArsR F2R1
pArsR F2R2
(-)
4

ArsR 1
ArsR 2
(-)
1 kb ladder

Expected Size
ArsR insert 600 bp

500 bp
5
ArsR in Backbone

Transformation efficiency highly improved by
using NEB cells instead of our own competent cells

6
Gold

Finally got golT out of Salmonella after using
genomic prep kit
Still haven't been able to get gesABC
going to order primers for gesABC proteins
individually instead of trying to get the entire
operon
Did a digestion/ligation yesterday of golT, golB
and golS
going to transform and plate them Monday since we
can't come in during the weekend

7
Gel of PCR
Expected Sizes golT 2300bp golB
200bp golS 500bp gesABC 6000bp
8
PCR - genomic and PCR purification template
We keep getting weird bands for gesABC and aren't
getting the correct 6kb band, so we are going to
order primers for ABC individually
9
Digestions - XbaI SpeI
10
Decoder

Update
Transformation efficiencies increased
significantly using NEB 5a and Turbo
electrocompetent cells.
Still searching for Gen5 software for HT Synergy
Plate Reader
Matt is working on
putting together a light
cannon

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15
Dippin' Dots Fundraiser
We had good sales Wednesday despite the rain,
selling 45 cups!