Team:Edinburgh OG/Design

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Design of the Golden Gate Destination Vectors for Transformation in R. jostii

We designed a set of Golden Gate destination vectors based on the MoClo standard to enable quick and easy way to create constructs and transformed it to our new Chassis, Rhodococcus jostii RHA1. We chose to use pSRKBB vector (Schmidt-dannert, 2014), which was derived from pSRK21 (Vesely et al., 2003) as the base of these destination vectors. This plasmid contains Corynebacterium replicon (pSR1) and has been successfully transformed to R. erythropolis, a closely related species of R. jostii (Knoppová et al., 2007; Vesely et al., 2003; Archer & Sinskey, 1993). It has also been refined by the removal of illegal sites incompatible with BioBrick standard (Schmidt-dannert, 2014). We removed the illegal sites (BsaI and BbsI) and replace the cloning site of the plasmid with a standard BioBrick cloning site flanked by two verification primer binding site (VF2 and VR), enabling colony PCR for quick screening of insert, derived from pSB1C3. The resulting vector (pSRKM_Empty) can be used as the template to make Level-1 destination vectors by digestion and ligation of standardized LacZα reporter flanked by fusion sites as described in Iverson et al. (2016).

Fig1. (A) Physical map of pSRKBB-Empty showing pSR1 replicon as a black band. The replicon contains per (a positive effector of replication) and rep (replicase) gene. pK19 backbone (white band) contains pBR322 ORI and Kanamycin resistance gene (KmR) regulated by the P45 promoter. Mutagenic primer binding sites are shown as red arrows (mut1, mut2, mut3, and mut4). The region in the red box was replaced by (B) a BioBrick cloning site flanked by standard verification primer binding sites, VF2 and VR, derived from Iverson et al. (2016), creating pSRKM_Empty. Destination vectors can be made by digestion and ligation with SpeI to insert standardized LacZα flanked by the designated Type IIs restriction enzyme (recognition sites shown as bold) and four base overhangs for Level-1 (C) or Level-2 (D) assembly. (E) Schematic map of the level-1 destination vector pSRKM_NN and (F) the level-2 destination vector pSRCM_NN with chloramphenicol resistance (CmR). X refers to the fusion sites and NNNN to its four base overhangs: A (GGAG), B (TACT), C (AATG), D (AGGT), E (GCTT), F (CGCT), G (TGCC), and H (ACTA).

References

Archer, J. A. & Sinskey, A. J. 1993. The DNA sequence and minimal replicon of the Corynebacterium glutamicum plasmid pSR1: evidence of a common ancestry with plasmids from C. diphtheriae. J Gen Microbiol, 139, 1753-9.

Iverson, S. V., Haddock, T. L., Beal, J. & Densmore, D. M. 2016. CIDAR MoClo: Improved MoClo Assembly Standard and New E. coli Part Library Enable Rapid Combinatorial Design for Synthetic and Traditional Biology. ACS Synthetic Biology, 5, 99-103.

Knoppová, M., Phensaijai, M., Veselý, M., Zemanová, M., Nešvera, J. & Pátek, M. 2007. Plasmid Vectors for Testing In Vivo Promoter Activities in Corynebacterium glutamicum and Rhodococcus erythropolis. Current Microbiology, 55, 234-239.

Schmidt-dannert, C. 2014. pSRKBB-empty (Plasmid #59449). www.addgene.org.

Vesely, M., Patek, M., Nesvera, J., Cejkova, A., Masak, J. & Jirku, V. 2003. Host-vector system for phenol-degrading Rhodococcus erythropolis based on Corynebacterium plasmids. Appl Microbiol Biotechnol, 61, 523-7.

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