Question

P. lac MYC coding sequence (fig73) amp ori You have successfully cloned MYC into the plasmid, as shown in the figure above. Using your understanding of plasmids and protein expression in bacteria, complete the table below:

Answer 1

Answer: In the given figure, coding sequence of the MYC gene is cloned under the control of P_lac promoter. Hence, the gene will express upon induction of promoter with IPTG only. Without induction, the gene will not express. IPTG (Isopropyl β- d-1-thiogalactopyranoside) is an inducer that will triggers the transcription of gene under the control of P_lac promoter.

The plasmid contain ampicillin resistant marker and ori site. The ampicillin resistant marker provides ampicillin resistant to bacteria that is transformed with this plasmid. Non-transformed bacteria will not grow on the ampicillin plate. Ori site is required for replication of plasmid in the bacteria. Without ori site, the plasmid will not replicate in the bacteria.

The below table represents the formation of colonies and expression of proteins in various scenarios

Condition	Colonies or no colonies	Protein expression or no protein expression	Explanation
Transformed with desired plasmid; grown on ampicillin; no IPTG added.	Colonies formed	no protein expression	The plasmid contain ampicillin resistant marker gene that provides ampicillin resistance to bacteria transformed with the plasmid. Hence, colonies will be formed. As there is no IPTG, Plac promoter is not induced and hence, MYC protein is not expressed.
Transformed with desired plasmid; grown on ampicillin; IPTG added.	Colonies formed	Protein expression	The plasmid contain ampicillin resistant marker gene that provides ampicillin resistance to bacteria transformed with the plasmid. Hence, colonies will be formed. As IPTG added, Plac promoter is induced to express MYC gene and hence, MYC protein is expressed.
Transformed with desired plasmid; grown on kanamycin (different antibiotic); IPTG added.	No colonies	no protein expression	The plasmid contain ampicillin resistant marker gene that provides ampicillin resistance but not kanamycin resistance to bacteria transformed with the plasmid. Hence, no colonies will be formed. As there is no colonies, protein will not express.
Transformed with plasmid; lacking ori; grown on ampicillin; IPTG added.	No colonies	no protein expression	Plasmids lacking ori site fails to replicate inside the transformed bacteria and hence, no colonies formed and no protein is expressed.
Transformed with plasmid; lacking Plac promoter; grown on ampicillin; IPTG added.	Colonies formed	no protein expression	The plasmid contain ampicillin resistant marker gene that provides ampicillin resistance to bacteria transformed with the plasmid. Hence, colonies will be formed. Although IPTG is added, Plac promoter is not there to initiate the protein expression. Hence, no protein is formed.