Team:Oxford/Improve

A fundamental aspect 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 fulfils the criteria of being:

  1. optimally expressed
  2. optimally secreted
  3. optimally effective against C.difficile.

Thus, we redesigned the 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 Salmonella typhimurium

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

  1. full-length form of the CD27L endolysin3
  2. SpyTag for purification and detection purposes
  3. Hexa-His tag for purification purposes

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 pipeline of:
  1. transformation of both constructs into E. coli (BL21 (DE3)-RIPL)
  2. miniprep + sequencing to verify successful transformation
  3. induction of expression with IPTG

However, despite identical growth and induction conditions, expression of the Dundee 2012 iGEM endolysin did not lead to protein induction that was detectable by Coomassie staining, even after testing on different days and triplicate independent structures, 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: SDS-PAGE Gel Comparing CD27L Endolysin Samples with Dundee-CD27L Samples

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: SDS-PAGE Gel of Truncated CD27L Endolysin1-179 Expression

As a further improvement, we decided to measure the killing efficacy of the CD27L_Assay endolysin on Bacillus subtilis (our surrogate target) compared to the truncated endolysin (as in the Dundee 2012 part), since no killing data was provided for the Dundee 2012 BBa_K895005 part. As seen in Figure 3, there is a substantially greater decrease in OD600 over time relative to the negative controls for the full-length CD27L endolysin when compared to the truncated, and thus, greater efficacy of the former.


Fig 3: Endolysin Killing Assay against B. subtilis and E. coli

References

# Reference
1 “Wet Lab Strategy.” Team:Dundee/Strategy, https://2012.igem.org/Team:Dundee/Strategy.
2 Mayer, M. J., et al. “Structure-Based Modification of a Clostridium Difficile-Targeting Endolysin Affects Activity and Host Range.” Journal of Bacteriology, vol. 193, no. 19, 2011, pp. 5477–5486., doi:10.1128/jb.00439-11.
3 Mayer, M. J., et al. “Molecular Characterization of a Clostridium Difficile Bacteriophage and Its Cloned Biologically Active Endolysin.” Journal of Bacteriology, vol. 190, no. 20, 2008, pp. 6734–6740., doi:10.1128/jb.00686-08.