Team:Groningen/Notebook/31 July 2009

From 2009.igem.org

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Wet

GVP Cluster

Transporters

GlpF

  • Restriction/ligase and transformation of GlpF in psB1AC3
Restriction
10ul GlpF PCR
2 ul Buffer
05.ul EcoRI
0.5 ul SpeI
7 uL MQ
  • 37 30min
  • Inactivation on Gel
  • Extraction from gel


Ligation
2 uL Ligase buffer 16° 1h → 65° 10 min
1 ul T4 Ligase
11 uL GlpF digested with EcoRI and SpeI 42° 2 min →
11 uL psB1AC3 digested with EcoRi and SpeI
Tranformation
  • add 4 uL of the GlpFpsB1AC3 ligation mixture to 50uL competent e.coli top10 cells.

Incubate:

  • 30 min @ ice
  • 5 min 37°
  • 5min @ ice
  • add 800uL LB
  • incubate for 1 h at 37°
  • plate on LB-AMP plates

Metal Accumulation

ArsR-MBP fusion

  • colony PCR MBP:
colony PCR MBP
Component amount
2x Phusion MM 12.5 uL
MQ 9.5 uL
MBP Fw 1 uL
MBP Rev 1 uL
Colony MBP 1 uL
Touchdown MBP program Temperature Time
Denaturing 98° 2.00 min
Touchdown 10X 60->50
Denaturing 98° 30 sec
Annealing 60°->50° 30 sec
Elongation 72° 30 sec
End cycles
Start Cycles 25X
Denaturing 98° 30sec
Annealing 60° 30 sec
Elongation 72° 30 sec
End cycles
Final elongation 72° 7 min
Hold Forever
  • Overnight culture MBP

Vectors

  • Metal sensitive promoters

Ligations were transformed into a new batch of competent cells and plated out on TY Amp100 plates.

  • Colonies of pSB3K3-HML and pSB1AC3-HML + RFP, and pSB3K3 or pSB1AC3 + pLac
    • pSB3K3-H, M, L + RFP all had colonies (not in all concentrations)
    • pSB1AC3-H, M, L + RFP all had colonies
    • pSB3K3 and pSB1AC3 + pLac had no colonies at all...
  • PCR to check pSB3K3-HML and pSB1AC3-HML + RFP
    • Pick 2 colonies per construct (put 1uL MQ in PCR tube and resuspend)
    • Boil 1 min @ microwave (max power)
    • Add MM and polymerase
    • Run programm: Colony
    • Expected size: 1420bp
    • Run products on 1% Agarose gel (unfortunately the gel was bad so the size could not be estimated)
    • Bands were seen for KH1&2, KM1&2, KL1, AH1&2, AL1.

Dry

We had a look at ways to measure things concerning gas vesicles (buoyant density, volume, etc.), buoyant density was previously measured using a specific apparatus which we're not likely to obtain. We also talked with some biologists about ways to detect protein concentrations (or rather, amounts) using antibodies, apparently this might be possible if we attach something for which there is a kind of "standard" antibody available (and the maltose binding protein we're attaching to ArsR might qualify as such).

To measure the total arsenic content in the cell you can use coupled mass spectrometry. for more details see Metal accumulation.


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