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CHARACTERISATION BY IIT MADRAS 2019
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BBa_K1825004 1
IIT-Madras
INTRODUCTION:
The cauliflower mosaic virus (CaMV) promoter 35S is a strong constitutive plant promoter (Odell et al. 1985; Benfey and Chua 1989). Studies indicate that this promoter can be used to express genes in E. coli.( Assaad and Signer 1990). This can be advantageous as expression can be verified rapidly and with a considerable saving of time and manipulation in many cases as this promoter is typically used with plant constructs.
We have validated the expression of an enhanced CaMV 35S promoter fused to a hph gene cassette in E. coli. This is a part of the plant vector pCAMBIA 1301 which has been used for our experiments. Details of the usage of this plasmid can be found here.
MATERIALS AND METHODS:
TOP 10 with the pCAMBIA 1301 plasmid was grown on LB agar. 100 ul of an overnight culture (5 mL) developed from a single colony was used to plate on 10 plates with varying concentrations of hygromycin (100-1000 ug/ml).
RESULTS:
E. coli had no visible growth in 50 ug/ml of hygromycin and this was found to be the minimum inhibitory concentration. This value is consistent with other studies.
Our transformed cells containing the hph gene conferring resistance to hygromycin B under the control of CaMV 35s was resistant up to 200ug/ml of hygromycin.
CALCULATIONS:
We calculated the initial number of cells to be 7.2 x 10^8 cells/ml based on the OD.
At 50ug/ml of hygromycin, the number of cells =7.2 x 10^8 cells/ml
At 100ug/ml of hygromycin, the number of cells=7.2 x 10^8 cells/ml
At 200ug/ml of hygromycin, the number of cells=(say 1/5th of what we had earlier) 9 x 10^7 cells/ml
Figure 11: %Sensitivity of cells in response to varying hygromycin B concentration
REFERENCES:
Odek J-T., Knowlton, s-, Li4 w., and Mauvais, C-J- (1988). Properties of an isolated transcription stimulating sequence derived from the cauliflower mosaic virus 35s promoter. Plant MOI. Biol. 10, 263-273.
Lam, E., Benfey, P., and Chua, N.-H. (1989a). Characterization of~~-l : A factor binding Site on the 35s pomoter of cauliflower mosaic virus. In Plant Gene Transfer: UCLA Symposia on Molecular and Cellular Biology, New Series, C. Lamb and R. Beachy, eds lNew York: Academic Press), in press.
Assaad FF, Signer ER. Cauliflower mosaic virus P35S promoter activity in Escherichia coli. Mol Gen Genet. 1990 Sep;223(3):517-20. PubMed PMID: 2176717.
Johnson AP, Burns L, Woodford N, Threlfall EJ, Naidoo J, Cooke EM, George RC. Gentamicin resistance in clinical isolates of Escherichia coli encoded by genes of veterinary origin. J Med Microbiol. 1994 Mar;40(3):221-6. PubMed PMID:8114074.
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