Team:Groningen/Notebook/27 August 2009

From 2009.igem.org

Igemhomelogo.png

Wet

GVP Cluster

DONE Check o.n. plates with transformed E.coli TOP10 cells
DONE Use colonies to grow o.n. precultures for plasmid isolation
TODO Test promoter strenght compared to BBa_J23101 promoter (Sven)
TODO Enter sequences of constructs to Sandbox

Colonies on Plates

Name Plasmid Used Antibiotics on Plasmid No. of Colonies Date
LAC-GVP (new) pSB1AC3 Ampicillin ~80 27/8
LAC-GVP (new, concentrated) pSB1AC3 Ampicillin ~200 27/8
LAC-GVP (old) pSB1AC3 Ampicillin 3 27/8
LAC-GVP (old, concentrated) pSB1AC3 Ampicillin 11 27/8
pBAD-GVP (new) pSB1AC3 Ampicillin 18 27/8
pBAD-GVP (new, concentrated) pSB1AC3 Ampicillin ~80 27/8
pBAD-GVP (old) pSB1AC3 Ampicillin 4 27/8
pBAD-GVP (old, concentrated) pSB1AC3 Ampicillin 28 27/8
pLow (constitutive)-GVP (old) pSB3K3 Kanamycin 0 27/8
pLow (constitutive)-GVP (old, concentrated) pSB3K3 Kanamycin 0 27/8
MQ (neg. control) X X 0 27/8
pHigh (constitutive)-RFP (pos. control) J61002 Ampicillin ~2000 27/8


→ The plates where old DNA fragments were used showed a lower amount of colonies, probably due to degradation of the DNA ends. In the case of the constitutive promoter BBa_J23109 no colonies at all were visible, and can be explained by both fragments being cut earlier.
→ From the "new" plates four colonies of each construct will be used for preculture growth, and from the "old" fragments only two will be used. (12 in total)

Transporters

HmtA

Colony pcr gave no positive response, samples 1.3.4.5.7.8.9.11 will be isolated for restriction check with NcoI that cuts in vector as wel as insert giving 2 fragments of 1814bp and 3261bp. An aditionnal control will be a pcr with VF2 and VR resulting in a fragment of 2222bp.

Below fisrt row 1-7, second row 8-12, F24,F2,F1

Clony-pcr-1-12,phusion,pBAD24,F1,F2.jpg

Metal Accumulation

FMT & MBP-ArsR colony PCR to check presence inducible promotors - 27august2009.JPG

1% Agarose (1xTBE) gel with Colony PCR reaction of fMT and MBP-ArsR with either low constitutive - or inducible Lac or Bad promotor. PCR was performed either using standard VR/VF primers to amplify entire region between prefix and suffix or the RBS oligo and fMT reverse primer. Expected size for fMT using VR/VF primers was ~500 bp and ~200 bp using fMT primers. MBP-ArsR should be ~1600 bp.

Vectors

Dry

April
MTWTFSS
    1 2 3 4 5
6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30
May
MTWTFSS
        1 2 3
4 5 6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 26 27 28 29 30 31
June
MTWTFSS
1 2 3 4 5 6 7
8 9 10 11 12 13 14
15 16 17 18 19 20 21
22 23 24 25 26 27 28
29 30
July
MTWTFSS
    1 2 3 4 5
6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30 31
August
MTWTFSS
          1 2
3 4 5 6 7 8 9
10 11 12 13 14 15 16
17 18 19 20 21 22 23
24 25 26 27 28 29 30
31
September
MTWTFSS
  1 2 3 4 5 6
7 8 9 10 11 12 13
14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30
October
MTWTFSS
      1 2 3 4
5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30 31
November
MTWTFSS
            1
2 3 4 5 6 7 8
9 10 11 12 13 14 15
16 17 18 19 20 21 22
23 24 25 26 27 28 29
30