Difference between revisions of "Part:BBa K3183201"

Latest revision as of 03:10, 22 October 2019

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 the bond between N-acetylmuramic acid and L-alanine¹ (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 N-terminal domain².

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

Figure 1: CD27L Catalytic Reaction.

Sequence and Features

Characterisation

We expressed endolysin in the pET28A vector following difficulties with Dundee's CD27L_1-179 endolysin BBa_K895005. We were able to achieve strong expression under the pET28A system (Fig. 3).

Figure 3: Fig. 2: SDS-PAGE Protein Purification Gel of CD27L_1-179 using Ni-NTA Affinity Chromatography.

Due to safety concerns, our endolysin killing assays could only be carried out on on Bacillus subitlis, a Category 1 bacteria as opposed to Clostridium difficile, a Category 2 bacteria. B. subtilis has a similar composition of peptidogylcan cell wall to C. difficile², allowing us to quantify killing data. We wanted to see if the truncated endolysin gave a measurable increase in lytic activity on B. subtilis as has been previously described in the literature². As seen in Figure 3, there is a substantial decrease in OD600 over time relative to the negative controls for CD27L, and a slight decrease in OD600 of B. subtilis for CD27L_1-179.

Figure 4: CD27L_1-179 Versus CD27L in Killing B. subtilis
The truncated CD27L_1-179 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.

Figure 4: Purification gel of CD27L_1-179 with SpyCatcher gel-shift assay. The purpose of this SDS-PAGE was to see after purification whether CD27L_1-179 is expressed and in the sample solution. The Spycatcher003-680 fluorophore can bind irreversibly to SpyTag by an isopeptide bond⁵, hence on the image we can see both SpyCatcher at around 19kDa and the SpyCatcher003-680 fused CD27L_1-179 at around 36kDa. Thus, we can confirm that our solution does contain CD27L_1-179.

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 CD27L_1-179 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 CD27L_1-179 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 CD27L_1-179 (BBa_K3183201). 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: CD27L_1-179 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: CD27L_1-179 Versus CD27L in Killing B. subtilis
The truncated CD27L_1-179 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. 5. Reddington, Samuel C., and Mark Howarth. “Secrets of a Covalent Interaction for Biomaterials and Biotechnology: SpyTag and SpyCatcher.” Current Opinion in Chemical Biology, vol. 29, Dec. 2015, pp. 94–99. DOI.org (Crossref), doi:10.1016/j.cbpa.2015.10.002.