Part:BBa_K4674011
The RepA and SSBP expressing cassette
This expressing cassette is regulated by lac operator. The N-terminal of SSBP protein is fused with a 6xHis-tag and a thrombin cleavage site.
The RCR mechanism: RepA protein, RCORI105 and RCORI65
Rolling Circle Replication (RCR) is a fundamental molecular process that plays an important role in DNA replication and various molecular biology applications. It involves one-way amplification of circular DNA samples, producing long single-stranded or double-stranded DNA products with repetitive sequences.
In RCR, each replication initiator enzyme (e.g. RepA) recognizes its corresponding double-stranded origin (DSO) of the target plasmid (e.g. pC194), and creates a nick on one of the DNA strands. The DNA polymerase uses an unnicked strand as a template to elongate the nicked strand. Finally, the elongated nicked strand is completely replaced by the newly synthesised strand, and the replication initiator enzyme will then ligate the elongated nicked strand into a circular ssDNA. The circular ssDNA could server as a template for secondard strand synthesis (Ruiz-Masó et al 2015).
Fig. The mechanism of RCR replication
The design of RCR mediated ssDNA production
To produce ssDNA, we decided to construct the RCR mechanism in E. Coli. We selected the replication initiator enzyme RepA, and the corresponding DSO, RCORI, from the target R-plasmid pC194 (Noirot-Gros et al., 1994). The last 105 nucleotides of pC194 DSO (RCORI-105) serves as the start point of RCR replication, while the first 65 nucleotides is applied as stop point. The cis-auto splicing sequence and tetrahedral ssDNA sequence are inserted between RCORI-105 and RCORI-65. For the activation of RCR, we cloned the RepA gene into pET15b, by which the protein expression could be regulated by lac operator. Furthermore, we insert another ribosome binding sequence (RBS) and the coding sequence of E. Coli ssDNA binging protein (SSBP) to protect the RCR-generated ssDNA. Finally, to purify the SSBP bound ssDNA, we fused a 6xHis-tag to SSBP.
Fig. The design of RCR mediated circular ssDNA generation
Experimental result
After cloning into the pET15b vector, We induced the RepA and SSBP protein expression by IPTG. After 6 hours of IPTG induction at 37°C, we found that the RepA and SSBP were expressed. However, the expression of protein was found in the inclusion body (cell pellet).
Fig. The coomassie blue staining of SDS-PAGE analysis of RepA and SSBP protein induction. S: supernatant; P: cell pellet.
References Ruiz-Masó J, et al. (2015) Plasmid rolling-circle replication. microbial spectrum.3;10.1128
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 47
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1230
Illegal XhoI site found at 1225 - 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 47
- 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 47
- 1000COMPATIBLE WITH RFC[1000]
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