Team:Groningen/Notebook/22 July 2009

From 2009.igem.org

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Wet

GVP Cluster

3A assembly of pSB1AC3, GVP, J23109, 106 and 100 (continued)

Transformation

  • 2 uL Ligation mix added to TOP10 competent cells from -80 stock
  • 30 min incubation on ice
  • 5 min heatshock 37°C
  • 5 min on ice
  • 950 uL LB added to eppendorfcup
  • 50 uL cells are added to the LB
  • Incubation: 1 hour, 37°C, waterbath shaker
  • 100 uL cells are streaked on LB agar plates (150 ug/ml Chloramphenicol)
  • Centrifugation: 1 min, 19000g
  • Pellet resuspended in 100 uL LB and streaked on LB agar plates (150 ug/ml Chloramphenicol)


Transporters

Metal Accumulation

Vectors

In order to check the VR VF2 primers that were ordered this year a gradient PCR was done with a vector that is expected to work pSB3K3, a pUC vector and a negative control with no vector.

VR-VF2
Component amount
MasterMix NH4 21 uL
VF2 iGEM09 1 uL
VR iGEM09 1 uL
vector 1 uL
Taq polymerase 1 uL


VR VF2 gradient program
Stage Temperature Time
Denaturing 95° 4 min
Start Cycles 30X
Denaturing 95° 1.30 min
Annealing Gradient 50°-62° 30 sec
Elongation 72° 1.30 min
End cycles
Final elongation 72° 6 min
Hold Forever

As a check-up we also did a restriction analysis for the pSB3K3 vector with EcoR1 and Spe1

double digestion
10x fast digest buffer 2 uL
pSB3K3 (22,5 ngr/uL) 8 uL (=180ngr up to 1ugr is recommended at fermentas)
EcoR1 1 uL
Spe1 1 uL
MilliQ 8 uL
Total 20 uL
  • Mix gently and spin down
  • incubate at 37° for 30 min.

Results of the PCR and restiction shown below. Bands were expected in the pSB3K3 lanes with VR-VF2 primers at 316bps. A band somewhat larger than 316 can be seen in lanes 2,3 (50°, 51.5°).

F102471 2009-07-22 07hr 04min ControlVRVF2 noted.jpg

The expected band for the digestion is 2727 bps, which is about the size of the band shown in the figure above. But there is no PCR product seen on gel, this might mean that the primers are not correct and at least that there seems to be no contamination of DNA in the primers. The PCR will be repeated.

  • Check VR/VF2 PCR products (21 July 2009) of pSB3K3 + promoters and pSB1AC3 + promoters on gel

F102471 2009-07-22 AC3-K3 vector.PNG


From left to right: 1kb marker (Fermentas), pSB3K3-promoter1 (low), K3-promoter2 (high), K3-promoter3 (med), pSB3K3 (control), pSB1AC3-promoter1 (low),AC3-promoter2 (high), AC3-promoter3 (med).

All show PCR product of ~300-350bp (except K3-3 which is very faint), but as the control shows no band it is not possible to say if the promoter insert can actually be found in the plasmid.

  • Number of colonies of inducible promoters transformants
transformants # colonies
pSB1A2 + R0010 (pLac) 30 + 50-70 on the concentrated plate
pSB2K3 + I0500 (pBAD) none

As the pSB2K3 + I0500 (pBAD)was not succesfully transformed, the transformation was redone.

  • Get more plasmid and single colonies for glycerol stocks

Grew o/n cultures of:

    • pSB1A2-R0010 (pLac) from 2 colonies in LB-Amp
    • pSB1AC3 with 3 promoters and pSB1AC3 empty in LB-Cam
    • pSB3K3 with 3 promoters and pSB3K3 empty in LB-Kan

Made single colony plates of:

    • pSB1A2-R0010 (pLac) from 2 colonies in LBA-Amp
    • pSB1AC3 with 3 promoters in LBA-Cam
    • pSB3K3 with 3 promoters in LBA-Kan

Grew o/n @ 37dg.

Dry

Jasper had a look at ArsD and Annelies at ArsAB, but at some point we realized that ArsA and ArsD might actually only be on some plasmids and may not be part of the genome of our E. coli (DH10B). To verify this we searched for a database which we could use for BLASTing (which we found) and BLASTed the sequences of several ars genes. The results (which can be viewed at Team:Groningen/BLAST) indicate that our E. coli most likely indeed does NOT have ArsA and/or ArsD, it most likely only has ArsB, ArsC and ArsR. This should save us quite a bit of work!

Today we worked on the efflux of arsenic by ArsA ArsB and ArsD. KB looked at ArsB first we looked at diffrent papers and we found some very promising ones In the afternoon we looked especially at As(III) and Sb(III) Uptake by GlpF and Efflux by ArsB in Escherichia coli by Yu-Ling Meng, Zijuan Liu, and Barry P. Rosen‡ [link] Escherichia coli* what it's influence is on the efflux of As and Sb, the influence of NADH, the influence of FCCP and the influences As and Sb on each other.


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