Part:BBa_K4673003

AmpR is a transcriptional regulator regulating the expression of the Citrobacter freundii ampC β‐lactamase gene. It belongs to the LysR family of transcriptional regulators that typically exhibits autoregulatory behavior by repressing its own expression (Ryuichi et.al, 2017). AmpR promoter serves as a critical element in the blue-white screening process, functioning as a binding site for RNA polymerase and leading to transcription of ampicillin resistance genes(β‐lactamase coding region). Generally speaking, it is difficult to discern whether each white E.coli DH5α colony comprises the plasmid with the target vector after conducting blue-white screening. However, ampicillin promoter and resistance help.

In our team’s project, we utilized the ampicillin promoter for the ampicillin resistance on the pGEM-T EASY vector as the second screening mechanism that can be used to confirm whether the surviving colonies in the results of blue-white screening took up the plasmid that contains the pGEM-T EASY vector during the transformation. In addition to the β-galactosidase marker, most cloning plasmids contain genes that confer resistance to an antibiotic such as ampicillin. Adding ampicillin in the culture medium should prevent bacteria that doesn’t contain the modified plasmid from growing, thereby offering evidence that the white colonies in the culture medium contain the plasmid with target vector. That is to say, the colonies with well-transformed vectors are considered to survive in the presence of ampicillin while the ones without could not function under the condition.