Question

53./The plasmid pFR55 is a useful v cloning of any restriction map of pFR55 s once or twice is s howing the restriction enzyme cutting sites for those endonucleases that cut the plasmid onl hown below. The plasmid can replicate in E. coli and carries the tetracycline resistance (Tc) and ampicillin resistance (Ap) genes for use as selectable markers in E. coli. Xmn Sall PstI Nrul Asel Tc BspMI BspMI RESTRICTION MAP OF pFR55 a. You wish to insert a gene into the Sall site of pFR55. How would you select for E. coli cells b. Name at least two other restriction enzymes which could be practically used for inserting a gene c. Is it possible to use antibiotics to determine whether pFR55 has received an insert in its EcoRI site? that have taken up the plasmid which contains the gene? into pFR55. Why, or why not?

The plasmid pFR55 is a useful vehicle for the cloning of any DBA sequence in E. coli. A restriction map of pFR55 showing the restriction enzyme cutting sites is shown below. The plasmid can replicate I'm E. coli and carries the tetracycline resistance Tc and ampicillin resistance (Ap) genes for use as selectable markers in E. coli.

a. you wish to insert a gene into the SalI site of pFR55. How would you select for E. Coli cells that have taken up the plasmid which contain the gene?

b. name as least 2 other restriction enzymes that could be practically used for inserting a gene into pFR55.

c. is it possible to use antibiotics to determine whether pFR55 has received an insert in its EcoRI site? why or why not?

Answer 1

Answer: a. In the pFR55 plasmid, restriction enzyme (RE) SalI falls within the sequence which codes for tetracycline resistance gene and hence when SalI site is used for inserting gene it disrupts the tetracycline resistance gene and it is no longer functional. So, in this case, we can select ampicillin antibiotic to screen for E.coli cells that have taken up the plasmid containing the insert in SalI site.

b. Restriction site selection for gene insertion depends on various factors such as insert should not contain selected restriction site within its sequence, directionality and so on. In the given pFR55 plasmid we can select RE based on the antibiotic resistance as the selection marker. Using ampicillin antibiotic while screening: RE pair-BamHI/BspMI or SalI/BspMI or EcoRI/BspMI, single RE-BamHI, BspMI. Using tetracycline antibiotic while screening: RE pair- EcoRI/AseI or XmnI/AseI, single RE-AseI, XmnI​.
c. Using the only EcoRI site for inserting gene won't disrupt ampicillin or tetracycline resistance gene and hence using antibiotics to screen for insert in EcoR1 site is not possible as it won't be able to differentiate between the cloned and plain plasmid.