Question

Copy of Shown below is a plasmid containing several labeled sequences. Multi-cloning site (CS) Transcription terminator Promoter TATA box original replication a) Based on the sequences it contains, is this plasmid intended for gene expression in eukaryotes or prokaryotes? How do you know? (3 pts) b) Based on your answer to part A, describe the promoter & terminator regions in a little more detail. What is a consensus sequence you would expect in the promoter? And what sorts of sequences should the terminator contain? (4 pts)

Answer 1

a) If we observe the plasmid diagram carefully, we can see that there is a promoter sequences present along with the TATA box and a transcription termination site a few base pair apart. The presence of promoter and transcription terminator site clearly say that when we will clone a transgene in between of these sequences, we can for sure express a transgene. Presence of the TATA box say that the promoter is a prokaryotic promoter, which means we can express the transgene inside the prokaryotic system.

B) The promoter sequence present preceding the gene, is a very important part for the expression of the gene. At the time of transcription, the sigma factor of the RNA polymerase, recognize the sequence present in the promoter to start expressing that specific gene by RNA polymerase.

There are two specific sequences present in the promoter which can be recognised by the sigma factors are -10 element or TATA box or Pribnow box and the another one is -35 element. These two sequences are consensus sequence and the distance between them is around 17-19 bases. The consensus sequence of the TATA box is usually 5'-TATAAT-3' and the consensus sequence of the -35 element is 5'-TTGACA-3'.

There are two different kind of transcription termination sites are present in the prokaryotic system. For the Rho dependent termination, there is a consensus rut site present. The sequence of the rut site not yet elucidated. For the Rho independent termination, a long poly(A) stretch is present in the termination site.

c) Inside the plasmid, a MCS or multiple cloning site is present. This site is specifically engineered to insert the transgenes in this location. You can also see that preceding the MCS, the promoter is present and the transcription termination site is present after the MCS, which make this ideal for cloning the transgene in the MCS region.

To clone the transgene inside the MCS region, you must first make the transgene construct ready which should contain two restriction endonuclease cut site in its two end ( you can do this by using specific primer in PCR process). Now, we will digest the plasmid and the transgene construct by two specific restriction enzymes ( which sequences had been introduced in the transgene construct) separately. Then we will let the digested products anneal with each other by mixing them in a tube followed by ligation. Now we have our recombinant product. ( The restriction enzymes used here should make staggered cut which will make overhang in the sequence and enable the annealing very smoothly.)