Coding

Part:BBa_K3183201

Designed by: David Schramm   Group: iGEM19_Oxford   (2019-10-16)
Revision as of 23:37, 19 October 2019 by Dschramm (Talk | contribs) (→‎Characterisation)


Truncated CD27L 1-179 with 6-His and SpyTag

This part contains CD27L fused to an N-Terminal 6-His Tag and SpyTag for purification, oligomerisation, and other SpyCatcher applications. This endolysin has previously been shown to be specific to C. difficile. This endolysin works by cleaving residues1 (Fig. 1), breaking the cell's peptidoglycan and compromising the structural integrity of its cell wall. Two X-ray crystal structures were solved for the N2. These are joined by a linker, which we've added ourselves (Figure 2).

Figure 2: X-ray Crystal Structure of the CD27L N-terminal domain (PDB codes 3QAY





Figure 1: CD27L Catalytic Reaction.












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]



Characterisation

We expressed endolysin in the pET28A vector following difficulties with Dundee's CD27L1-179 endolysin BBa_K895005. We were able to achieve strong expression under the pET28A system (Fig. 3).

File:BBa K3183012 Purification Gel.png
Figure 3: Fig. 2: SDS-PAGE Protein Purification Gel of CD27L1-179 using Ni-NTA Affinity Chromatography.

To confirm the identity of our endolysin, we wanted to run it through electrospray mass spectrometry (ESI-MS). However, given that we didn’t have access to size-exclusion chromatography, we were only able to send our CD27L protein preparation, as the other sample was far too dirty for effective ESI-MS. The identity of our endolysin, as well as putative autocleavage is shown and discussed in Fig. 4.

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 BBa_K895005 part submitted by the 2012 Dundee iGEM team, which consists of:
1. truncated form of the CD27L endolysin, shown to increase both efficacy and host range2
2. Type VI secretion protein derived from S. typhimurium
3. Hemagglutinin (HA) Tag for Western Blotting

In contrast, our novel part BBa_KK3183200 (CD27L) 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:
1. transformation of both constructs into E.coli (BL21 (DE3)-RIPL Competent E.coli)
2. miniprep + sequencing to verify successful transformation
3. induction of expression with IPTG

Figure 1.1: Dundee CD27L1-179 Construct in pET28A
Figure 1.2: 6His-SpyTag-CD27L Construct in pET28A



Thus, we sought out the endolysin in an existing part made by the 2012 iGEM Team Dundee. 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.

Figure 2: Dundee CD27L1-179 Expression Gel



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. We added 6His and SpyTag to the N-terminus to allow for detection (via SpyCatcher gel-shift assays) and purification. This resulted in BBa_K31830201 (CD27L1-179</sib<). 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.

Figure 3: CD27L1-179 Purification Gel



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.

Figure 4: CD27L1-179 Versus CD27L in Killing B. subtilis
The truncated CD27L1-179 shows decreased growth relative to the negative control; however, log-phase growth resumes after 150 minutes. CD27L endolysin results in decreased growth during the first 100 minutes, and growth thereafter appears to be linear. This may point to inhibited log-phase growth in subsequent generations of B. subtilis following CD27L exposure.

Reference

1. 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.
2. Mayer, M. J et al. “Structure-based modification of a Clostridium difficile-targeting endolysin affects activity and host range.” Journal of bacteriology vol. 193,19 (2011): 5477-86. doi:10.1128/JB.00439-11
3. Dunne, Matthew et al. “The CD27L and CTP1L endolysins targeting Clostridia contain a built-in trigger and release factor.” PLoS pathogens vol. 10,7 e1004228. 24 Jul. 2014, doi:10.1371/journal.ppat.1004228
4. Twetman, Svante, et al. “Scanning Electron Microscopic Study of Streptococcus Mutans BHT Lysed by Lysozyme.” European Journal of Oral Sciences, vol. 93, no. 1, 1985, pp. 23–29., doi:10.1111/j.1600-0722.1985.tb01304.x.

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