Team:Tokyo-Nokogen/Project/Lysis

[Introduction]
Traditional protein purification procedures rely on sonication or French press for cell disruption to liberate the expressed protein. The novel protein purification procedure, ESCAPES (Escherichia Coli Auto Protein Synthesizer), proposed by our team attempts replace this tiresome step by employing a phage lysis system. In our ESCAPES system, after cellular aggregation and decantation, the holin and endolysin genes are induced to disrupt the cells, thus conveniently liberating the expressed protein.

[Objective and Parts]
We attempted to construct the series of parts described below (A~C). We did not know whether the SRRz genes were preceded by RBS, so we attempted to construct both A and B.

[Method]
1. Construction of the parts (A~C)
Following the protocols of the BioBrick™ Assembly Manual (http://ginkgobioworks.com/support/BioBrick_Assembly_Manual.pdf), we constructed is this order:

1.[SRRz-Double terminator (ST)] and [S105RRz-Double terminator (S105T)]

2.[RBS- SRRz-Double terminator (RST)] and [RBS-S105RRz-Double terminator (RS105T)]

3.[pBad/araC- RBS- SRRz-Double terminator (pRST)]

The plasmid containing pRST was evaluated by the plate assay in which DH5a/pSB1k3-pRST were induced by 2.5 mM L-arabinose.
2. Revalidation of the standard biological parts (SRRz and S105RRz).
We analyzed the sequences of the registered standard biological parts SRRz (BBa_K124003) and S105RRz (BBa_K124017). We also tried to amplify the genes encoding SRRz and S105RRz by PCR with forward and reverse primers.

[Results and Discussion]
1. Construction of the genes (A~C)
We could only construct pRST (A). However, we could not confirm the lysis function by the plate assay. And a part of pRST, RST digested by EcoRІ and PstІ did not have SRRz (Fig. 1). Instead, it contained a 400 bp-long fragment. We therefore believe the registered standard biological parts BBa_K124003 and BBa_K124017 do not contain the expected inserts, which are described in the registry.