iGEM%20Bioware

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iGEM Bioware

• Week 9

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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. 

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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

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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
•  
•  

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Gel of PCR
Expected Sizes golT 2300bp golB
200bp golS 500bp gesABC 6000bp
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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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Dippin' Dots Fundraiser
We had good sales Wednesday despite the rain,
selling 45 cups!

• Selling again this afternoon from 1230-330 in
  front of the Union