Part:BBa_K2442372

Bacteriophage T7 lysozyme (LysS) coding sequence

LysogenS (from the LysS plasmid) is a T7 bacteriophage protein which acts to repress T7 polymerase synthesis, and facilitate lysis of E. Coli cells. This was used to maintain non-toxic expression of a protein under control of a T7 promoter, which could potentially become toxic if overexpressed.

T7-Inducible System and Lysozyme

The inducible T7 expression system is a highly controlled system for overproduction of proteins in E. coli. BL21(DE3) carries a chromosomal copy of the enterobacteria phage T7 RNA polymerase gene expressed from an Isopropyl β-D-1-thiogalactopyranoside (IPTG) inducible lac promoter. T7 RNA polymerase initiates transcription exclusively at T7 promoters. Therefore, a plasmid-borne gene placed downstream of a T7 promoter and an efficient ribosome binding site (RBS) will be highly expressed in an IPTG-inducible fashion in this strain. As well as IPTG induction of RNA polymerase, two other regulatory mechanisms can be used to fine-tune expression levels: Lac operator sites can be incorporated into the T7 promoter to repress transcription in the absence of IPTG, and the lysozyme protein of T7 (encoded by T7 gene 3.5, but referred to as LysS) inhibits transcription by T7 RNA Polymerase. Expression of lysozyme has been used extensively to repress basal transcription of the gene in the absence of IPTG to avoid toxic effect of some proteins. Repressing basal expression in the absence of IPTG helps to stabilise expression plasmids, and guard against selections for mutations in the T7 promoter or gene coding region. Two T7 lysozyme expression plasmids are commonly used: pLysS expresses lysozyme at low levels, giving better induced expression levels but slightly higher leakage than pLysE which expresses lysozyme at higher levels

Regulatory Plasmid

The T7 lysozyme gene was isolated from pLysS (where it is called T7 gene 3.5, but referred to here as LysS) using colony PCR, and was then ligated in to pSB1C3 initially, although the final plasmid would have used pSB3K3. This is because all biobricks are submitted in pSB1C3, and also to avoid potential competition between plasmids, which can arise if both the expression and regulatory plasmids have the same antibiotic resistance or the same origin of replication.

pLysS was used as template DNA in the 50µl colony PCR reaction, where 3 reactions were carried out using three different colonies, so as not to choose a single mutated colony. Primers were designed (containing biobrick prefix and suffix sequences) to flank the 456 nucleotide lysS gene for amplification (Table 3). Following PCR, the PCR product was digested with EcoRI and PstI to prepare for ligation in to pSB1C3 (digested with the same restriction enzymes). Following the ligation reaction, the plasmids were then transformed and cloned in DH5α cells. To ensure the plasmids were correct, the plasmid was isolated, digested and observed in a gel: a 3ml overnight culture was made using transformant colonies, which were left overnight to grow. The culture was then miniprepped and digested with EcoRI and PstI, before being ran on a 2% agarose gel electrophoresis. The band sizes correlated with the expected vector and insert sizes, indicating successful transformation, and this was later confirmed by sequencing (Figure 5).

Furthermore, a constitutive promoter would have been added in front of the lysS gene using PCR: Primers were designed to add a variety of promoter with different strengths before the gene, and thus we could select the lysozyme transcription level with the greatest transformation success. However, lysS was only successfully ligated in to pSB1C3, with no promoter.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]