Problem statement: It is widely accepted thought that the weak promoters
control the RNA synthesis and play regulatory role in complex genetic networks
in bacterial system. An experiment had been designed to address whether mutations
in the -16/-17 region affect the rate of transcription at an activator-independent
promoter in E. coli or not?
Approach: The aim of this study was to determine whether mutations in the
-16/-17 region affect the rate of expression at an activator-dependent promoter
in JM109 strain of E. coli. Primers were constructed to amplify the mutant
promoter genes through PCR. The amplified PCR product was checked and then inserted
into the MCS region of pAA128 plasmid. Further the plasmid vector was transformed
into JM109 strain of E. coli and then cloned the selected transformats.
Finally, the plasmid from each mutant colony was then sequenced using the protocol
supplied with the Amersham Pharmacia Biotech T7 sequencing Kit. The JM109 cultures
for which the sequences were determined, then assayed for β-galactosidase
activity to assess the rate of gene expression from the altered promoters.
Results: The present investigation revealed that the extended-10 promoter
region has a substantial effect on the rate of transcription at weak promoter
sequence and also bearing little resemblance to the consensus sequence recognized
by RNA. The expression of the genetically engineered plasmid proved that the
2 bps (-16 and -17 base pair) found adjacently upstream of the extended-10 promoter
have an effect on the level of transcription. This was achieved by site specific
base substitutions into the weak promoter of a modified lac operon lacking any
activator or repressor binding sites. The results from gene expression assays
of several mutants showed a distinct preference for either GG or TT located
adjacently upstream of the extended promoter element. Thus the present study
emphasized that extended promoter region also played a key role in regulation
transcription initiation in JM109 strain of E. coli.
Conclusion: The present study concluded that the site specific changed in
the extended promoter regions, particularly the-17/-16 base pairs had greater
influence in the transcription initiation in E. coli. Thus the promoter
engineering study will definitely pave the way to do both, on or off the genetic
switches in bacterial system according to our needs to produce high protein
of interest or decrease or block the expression of a particular unwanted protein.