Difference between revisions of "Part:BBa K3183200"

Revision as of 21:05, 19 October 2019

CD27L with N-Terminal 6-His Tag and SpyTag

CD27L with N-Terminal 6-His Tag and SpyTag

Sequence and Features

Characterisation

Part Improvement of BBa_K895004

A fundamental crux of the ProQuorum system is the expression of the endolysin which is responsible for the actual lysis and consequent killing of the pathogenic C.difficile . Thus, there is a clear need for an endolysin that fulfills the criteria of being:
1. optimally expressed
2. optimally secreted and
3. optimally effective against C.difficile

Thus, we redesigned the [parts.igem.org/BBA_K895005 BBa_K895005] part submitted by the 2012 Dundee team1 , which consists of:
1. truncated form of the CD27L endolysin, shown by Mayer et al. (2011)2 to increase both efficacy and host range
2. Type VI secretion protein derived from S. typhimurium
3. Hemagglutinin (HA) Tag for Western Blotting

In contrast, our novel part BBa_K3183009 (CD27L_Assay) encodes:

1. full-length form of the CD27L endolysin
2. SpyTag for purification, oligomerisation, and other SpyCatcher applications
3. 6-His tag for easy purification

Incorporating both of these parts into pET28A, our expression vector, we planned on testing the relative efficacies of B.subtilis killing as per our killing assays. Thus, we followed our generic pipeline of:
transformation of both constructs into E.coli (BL21 (DE3)-RIPL Competent E.coli)
miniprep + sequencing to verify successful transformation
induction of expression with IPTG
Dundee Endolysin vs Normal Endolysin
However, despite identical growth and induction conditions, expression of the Dundee 2012 iGEM endolysin was unsuccessful, even in triplicate, as shown in Figure 1. In contrast, expression of the CD27L_Assay part was successful and quantitatively verified via both mass spectrometry and BCA assay, as in the Results page.
Fig 1 Thus, to solve this issue, we decided to attempt expression of only the truncated endolysin from the Dundee 2012 Biobrick, without its substantially large Type VI secretion tag. This resulted in BBa_K31830012 (CD27L_Truncated 1-179). Upon analysis, there is evident expression of the truncated endolysin as seen in Figure 2. However, given identical conditions of growth and induction, there appears to be greater expression of the full-length endolysin, which may perhaps indicate greater stability.
Fig 2.
As a further improvement, we decided to measure the killing efficacy of the CD27L_Assay endolysin on B. subtilis (our surrogate target) as no killing data was provided for the Dundee 2012 BBa_K895005 part. As seen in Figure 3, there is a substantial decrease in OD600 over time relative to the negative controls.
Fig 3.