Team:Groningen/Notebook/20 July 2009

From 2009.igem.org

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Wet

GVP Cluster

Precipitation of the restriction fragments from GVP, J23109, J23100 and J23106

To concentrate BBa_J23109, BBa_J23100, BBa_J23106 and GVP it is first precipitated and then MQ is added.

Procedure:

  • 100 μl absolute ethanol is added to ~50 μl Restriction fragment of GVP (12.1 ng/μL),J23109 (13.1 ng/μl), J23100 (11.9 ng/μl) and J23106 (11.1 ng/μl).
  • Incubation -80°C for 1 h
  • Centrifugation 30 min 0°C (14000rpm)
  • Supernatant is removed
  • Wash with 1000 μl 96% Ethanol (added ethanol and inverted a couple of times)
  • Centrifugation 10 min 4°C (14000rpm)
  • o/n air dried on bench top


Preparation of 1-14N and 1-1D for glycerol stocks, restiction analysis and 3A assembly

1-14N (BBa_I0500) and 1-1D (BBa_R0010) are inducible promoters, induced by L-arabinose and lactose (or IPTG) respectively.

  • DNA from iGEM plates resuspended in 15 μL MQ and stored at -20°C


Transporters

Below the PCR of 17/07, There seems to be a vague band at ~1150 in lane 1.


F102471 2009-07-17 08hr 29min pcr1,pcr2 noted.JPG

The band at ~1150 kb was cut out of the gel and used for pcr described below.

PCR1 gel
Component amount
MasterMix NH4 40 uL
HmtA Fw 1 uL
HmtA mut1RC 1 uL
PCR1 band
Taq polymerase 2uL
PCR1 program Temperature Time
Denaturing 95° 5 min
Start Cycles 30X
Denaturing 95° 20 sec
Annealing 55° 20 sec
Elongation 72° 1.10 min
End cycles
Final elongation 72° 10 min
Hold Forever

Metal Accumulation

Vectors

Positive control for the psB1AC3 vector done with both new and last years primers.

VR-VF2
Component amount
MasterMix NH4 21 uL
F primer 1 uL
R primer 1 uL
psB1AC3 vector 0.5 uL
Taq polymerase 1 uL


Primers
Cup nr F primer R primer
1 VF2 iGEMGr09 VR iGEMGr09
2 VF2 iGEMGr09 VR iGEMGr08 Aug
3 VF2 iGEMGr09 VR iGEMGr June
4 VF2 iGEMGr08 VR iGEMGr09
5 VF2 iGEMGr08 VR iGEMGr08 Aug
6 VF2 iGEMGr08 VR iGEMGr June


VR VF2 program
Stage Temperature Time
Denaturing 95° 5 min
Start Cycles 30X
Denaturing 95° 20 sec
Annealing 61° 20 sec
Elongation 72° 20 sec
End cycles
Final elongation 72° 10 min
Hold Forever

The gel shown below shows bands at about 1000 which is not what would be expected. Band of 315 were expected for lanes 1-6. --> contains ccdB gene, which would increase the expected fragment size to 991bp! This is about the size which is seen on gel....

F102471 2009-07-20 08hr 30min noted.JPG

vectors

Colony PCR on vectors for checking if promotors were inserted

- expected lengths (primer VF2 & VR) on:

psb1AC3 316 bp




Dry

Jasper looked at GlpF import, trying to create a prototype model in Simbiology by playing with the parameters. However, it turns out that Simbiology doesn't allow reactions using reactants from different compartments, so using a Michaelis-Menten equation (instead of using more fine-grained reactions) is pretty much the only option for transporters. Also, after discovering that it is quite hard to directly derive the required constants from the given graph manually (due to the different capacities of the cell and the solution) a table was made of the uptake graph (Fig. 1B) in Meng2004 by importing the graph in Inkscape and aligning the axes with its rulers.


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