Difference between revisions of "Part:BBa K4083000"

Latest revision as of 00:59, 22 October 2021

pRGPDuo2 plasmid for P. putida

Duo2 plasmid is engineered by Rahul Gauttam, Aindrila Mukhopadhyay, and Steven W. Singer (2020)[1]. The plasmid was sent to us by Rahul Gauttam who is currently working as a postdoctoral fellow at Berkeley Lab. This plasmid was modified from the pRG_Duet vector for Corynebacterium glutamicum.

Usage and Biology

Figure 1. Illustration of Duo2 plasmid.

As illustrated in Figure 1, this plasmid contains two multiple cloning sites (MCS 1 and MCS2) with two transcriptional terminators. The MCS1 and MCS2 are regulated by IPTG-inducible Ptac and aTc-inducible PtetR/tetA promoters respectively. Moreover, there are lacI and tetR dependent repression systems that are regulated by Ptac and PtetR/tetA respectively. They were integrated for metabolic engineering purposes. There is a kanamycin resistance gene (KanR). There are two origins of replication to allow replication in E. coli (colE1) and P. putida (pRO1600).

Our team incorporated the rhla/rhlb and nadE genes into MCS1 and MCS2 respectively. Our team planned to extract rhLA and rhlB genes from P.aeruginosa and to insert them into pRGPDuo2 plasmid. We developed the new approach to increase the P. putida's rhamnolpid synthesis by adding nadE gene which encodes NAD synthetase. This way, we hoped to see more rhamnolipid production in engineered P. putida.

Part functionality

Reference

[1] Gauttam, R., Mukhopadhyay, A., & Singer, S. W. (2020). Construction of a novel dual-inducible duet-expression system for gene (over)expression in Pseudomonas putida. Plasmid, 110. https://doi.org/10.1016/j.plasmid.2020.102514

Sequence and Features