Coding

Part:BBa_K3183200

Designed by: David Schramm   Group: iGEM19_Oxford   (2019-10-16)


CD27L with N-Terminal 6-His Tag 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 (Fig. 2) structures were solved for the N-terminal2 and C-terminal domains3.

Figure 2: X-ray Crystal Structure of CD27L; PDB codes 3QAY (green) & 4CU5 (purple) with a linker (yellow) drawn in with PyMol by Team Oxford 2019. Note the Zn2+ ion (silver) present in the putative active-site cleft, which may be involved with catalysis. The domains are joined by a linker, which we've added ourselves.





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).

Figure 3: SDS-PAGE Protein Purification Gel of CD27L using Ni-NTA Affinity Chromatography.

To confirm the identity of our endolysin, ran 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.

Figure 4: Mass Spectrometry of CD27L Endolysin Following Ni-NTA Purification (Corresponding amino acid positions in brackets.)

Due to safety concerns, our endolysin killing assays could only be carried out on Bacillus subitlis, a Category 1 bacteria as opposed to Clostridium difficile, a Category 2 bacteria. B. subtilis has a similar composition of peptidoglycan (A1 γ type) cell wall to C. difficile2, allowing us to quantify killing data.

To get more information about the killing kinetics of the CD27L endolysin, we titrated our killing assay with increasing concentrations of CD27L endolysin. This titration showed measurable effect on B. subtilis cultures in LB broth with final endolysin concentrations greater than 0.2mg/mL (Fig. 5). Rate constants regarding how much the endolysin inhibited growth were subsequently used to inform our model (Table 2).

From this data, we were able to obtain kinetic constants for endolysin killing, which were subsequently used in our mathematical models.

Figure 5: CD27L Endolysin Killing Assay Varying Final Endolysin Concentration; Here we show that the effect of the endolysin appears to inhibit growth at concentrations above 0.002mg/mL. Growth seems to increase significantly at 150 minutes, which may be indicative of CD27L degradation.Interestingly, 0.2mg/mL of endolysin grows linearly for duration of the assay (over 6 hours), as does 0.45mg/mL after 150 minutes. This may mean that log-phase growth is inhibited under these conditions.

We also showed that endolysin was specific to B. subtilis using a killing assay involving E. coli, B. subtilis, and our chassis Lactobacillus reuteri (Fig. 6 & 7).

Figure 6: CD27L Endolysin Killing Assay on E. coli versus Bacillus subtilis; The above graph demonstrates the specificity of the CD27L endolysin, by assaying killing on E. coli, the most common chassis used in synthetic biology applications.
Figure 7: CD27L Endolysin Killing Assay on Bacillus subtilis versus Lactobacillus reuteri; While L. reuteri grows to a smaller maximum OD600 than B. subtilis, there is no discernable change in OD600 due to endolysin . Notably, the effect of lysozyme on L. reuteri is relatively small due to its thick bacterial cell wall, and we were unable to show a killing effect. For this reason, ampicillin was chosen as our positive control.




















Finally, we fortunate enough to obtain scanning electron microscopy (SEM) images thanks to Errin Johnson at the Dunn School of Pathology. After performing our endolysin killing assay, we fixed the cells onto glass slides and imaged them. We chose this as a qualitative method to determine if CD27L had a similar effect on endolysin killing as lysozyme4. Below shows images of B. subtilis following CD27L endolysin treatment (Fig. 8). Here, we demonstrated some changes in B. subtilis cell morphology, but further investigation is needed to explore these effects.

Figure 8: Scanning Electron Microscopy (SEM) Images of B. subtilis after 60 Minute Incubation with CD27L Endolysin versus Negative Control; colony morphology in the endolysin group appears to contain small spots, as well as cell deflation. While the latter may be due to sample fixation, the spots may be indicative of structural compromise in the cell wall. This is believed to be due to compromised structure of the peptidoglycan scaffold, resulting in osmotic lysis. This is likely responsible for the blebs and debris notable especially in Fig. 11. These changes are not local to one or two cells but extend over large regions of the culture (Fig. 10). Moreover, the disruptions in the membrane integrity following endolysin treatment are also indicative of the early stages of lysis. These blebs and disruptions are much more stark when viewed against the control. However, these observations will have to be explored further to definitively confirm the effects of CD27L endolysin on B. subtilis morphology, as lysis does not appear to be as stark as with lysozyme4.

Part Improvement of BBa_K895005

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</b>

In contrast, our novel part BBa_K3183200 (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 2. 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). Upon analysis, there is evident expression of the truncated endolysin as seen in Figure 3.1. However, given identical conditions of growth and induction, there appears to be greater expression of the full-length endolysin (Fig. 3.2), which may perhaps indicate greater stability.

Figure 3.1: SDS-PAGE Protein Purification Gel of CD27L using Ni-NTA Affinity Chromatography.
Figure 3.2: SDS-PAGE Protein Purification Gel of CD27L1-179 using Ni-NTA Affinity Chromatography























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 4, 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.

References

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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