Template:Team:Edinburgh/Notebook/Week03

1) Cloning of ccm PCr product into vector

pSB1C3 vector with RFP was used
PCR product was cloned using modified Cfrench:bbcloning protocl
reaction mixture:
water 30 ul
vector pSB1C3 5 ul
PCR ccm 5 ul
buffer 3 5 ul
BSA 1 ul
XbaI 2 ul
PstI 2 ul
components were mixed, pulse-spinned in a centrifuge and incubated for 3 hours in 37 oC

1)Transformation of Citrobacter with various replicons (results)

These results show that all but one of the plasmids have successfully been transformed into both E. coli and Cf, therefore these replicons are compatible with Cf. Cf cells with the multi-host plasmid did not grow at all, and there can be several reasons why: the codon usage in Cf differs from E. coli and Bacillus; the promoter does not work properly in Cf or Cf is less resistant to chloramphenicol. Bacteria transformed with ptg262 will be plated onto plates with differing concentrations of chloramphenicol to assess whether this plasmid is compatible at all with Cf.
The pSB2K3 transforms were plated onto plates containing X-gal in order to assess whether the Cf cells were lacZ positive (had a lacZ gene on their chromosome). Since the colonies turned blue, this means that the Cf cells are lacZ positive (as the plasmid contained no lacZ minigene) whereas the E. coli cells are lacZ negative since the colonies were red. The RFP was expressed in both cells, as evidenced by red fluorescence under a blue light.
2)LA agar plates with different metronidazole and DNBA concentration were examined.
No IPTG was added and no difference was seen between the strains. However, 50 ul (0.1 mg/ml) DNBA/met was chosen as further concentration to use since it did not seem to inhibit growth of the strains substantially.
3)Repeating LA agar plates with metronidazole, nitrofuratoin and DNBA.
50 mg/ml stock of nitrofuratoin ( antibiotic toxic to E.coli) was prepared.
Plates with 0.1 mg/ml DNBA, met or NFT plus and minus IPTG were prepared and inoculated with the four nitroreductase strains.
4)Test if sucrose hydrolase can act as arsenic detector
Chloramphenicol (5 ul out of 40 mg/ml stock), sucrose ( 250 ul out of 20% sucrose stock) and 0/5/25/50 ul As (out of 10 000 pps As stock) were added to LB (5 ml). The four bottles were inoculated with pSBIC3-J33207 CScA blue (4)

5) PCR for MtrCAB and ccmA-H
Stock solutions of primers were prepared by adding water to fresh primers in order to reach 500 pmol/ul concentration.
Working solution was prepared by adding water to stock solution in order to reach 10 pmol/ul concentration.
Cell suspensions of E. coli and S. oneidensis were obtained by inoculating cells in 150 ul water
PCR mixture was prepared according to Cfrench:PfuPCR protocol and PCR was performed for total of 100 minutes.
In the meantime agarose gel was prepared following Cfrench:AGE protocol
PCR product was tested on the gel resulting in clear band of about 6 kb for ccm and no bands for Mtr


ccm PCR product was purified following Cfrench:DNAPurification1 protocol . Purified product was left in freezer for storage [freezer 1 (B grade freezer, the leftmost one), orange rack on top shelf, 1st row, 5ft tube markes KK 9.7.12 ccm PCR E. coli]