Team:Edinburgh OG/Proof

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Demonstration of the E. coli - Rhodococcus Golden Gate Vectors

A. Demonstration of Multipartite Assembly

To demonstrate the functionality of the E. coli - Rhodococcus Golden Gate destination vectors for multipartite assembly, the destination vectors was used to assemble level-0 parts into a transcriptional unit.

Fig1. Demonstration of the MoClo assembly using the developed vector. (A) Single transcriptional unit assembly, and (B) combinatorial assembly of three coding sequences with varying size enables quick screening by colony PCR for demonstration.

Assembling single transcriptional units with destination vector pSRKM_AE showed a high assembly efficiency, with 92% white (non-blue) colonies (Table 5), where 8/8 colonies have correct insert (Figure 15). Transformed colonies were grown in liquid culture overnight and showed expression of the red fluorescent protein (Figure 14A)

Even though the construct has the same promoter and RBS as the RFP construct, no expression was observed (Figure 14 B, C, D). The same promoter and terminator, and also the same vector backbone were used, suggesting that failure of expression can be caused by either the coding sequence or the fusion sites (E and F). As the coding sequence used (except for LacI) are a well-characterized reporter gene, the later are probably the cause.

Fig1. (A) pSRKM_AE – RFP, (B) pSRKM_EF - phiLOV, (C) pSRKM_EF - eBFP, and (C) pSRKM_EF - LacI. Only construct pSRKM_AE – RFP showed expression of the reporter protein.

B. Transformation to Rhodococcus

The destination vectors and its derivative was transformed to R. jostii with the method modified from Kalscheuer et al. (1999) and plated on Kanamycin plates with X-gal and IPTG for selection and screening. As different construct inserted into the vector might affect transformation efficiency, the empty vector pSRKM_Empty was transformed along with pSRKM_AE (contain LacZα), pSRKM_RFP (J23100-B0034-RFP-B0015), and compared to the original backbone, pSRKBB. All transformed R. jostii were growing in Kanamycin plates while untransformed plate does not show any colonies growing. They also showed less efficiency than the previously described, ranging from 33 – 360 CFU / ng DNA.

Fig1. (A) Colony PCR of the Rhodococcus cells transformed with pSRKBB, pSRKM_Empty, and pSRKM_AE. (B) Positive control and colony PCR of the Rhodococcus cell transformed with pSRKM – RFP. A faint band was shown for colony harbouring pSRKBB as it only has one verification primers binding site, also seen in positive control. Other pSRKM derived vectors showed a clear band with correct sizes, also confirmed with the positive control. Colony 1 of the pSRKM_RFP does not show any band which would be likely caused by technical errors.

Colony PCR using the standard verification primer (VF2 and VR) was used to further confirm transformation of the plasmid into R. jostii. All destination vector developed in this study have the binding sites for VF2 and VR primer, while the pSRKBB only have one binding site for VR primer. Positive bands were showed by the destination vectors, but not for pSRKBB (Figure 17). This suggest that the destination vectors we created has been successfully transformed to R. jostii.