Team:Oxford/Experiments
Experimentation
Our experimentation with E. coli involved many different techniques which are detailed below:
For our project, we designed several constructs which were used for various experiments. To assemble these constructs, we used Gibson assembly.1
The Gibson assembly offers a quick and easy method to construct DNA fragments and recombinant plasmids. DNA with overlapping ends are added Gibson assembly master mixes, which contain T5 exonuclease, Taqligase, and Phusion DNA polymerase1. Fragments are then assembled all together in a 4-step process (Fig. 1).
Figure 1. Overview of Gibson Assembly. (Gibson Assembly by NivinN is licensed under CC BY 4.0)
Despite the Biobrick standard being based around restriction enzymes, we used Gibson Assembly to construct all of our recombinant plasmids. Having our main inserts synthesized by IDT, we only needed to clone the fragment into the corresponding vector. This was achieved by creating overhangs using appropriate primers in polymerase chain reaction (PCR) in the inserts and the vector backbones. The resulting fragments were separated by agarose gel electrophoresis to purify only the bands corresponding to our fragment of interest. The purified DNA was then used for Gibson Assembly, which linked the fragments together, creating the final recombinant plasmid constructs.
Moreover, this method enabled us to easily troubleshoot our assemblies simply by ordering new primers. We encountered such issues, notably when we needed a construct with only a promoter and a reporter gene (e.g. erm-mClover3, ldh-mClover3) or when terminators made large IDT gBlocks difficult to synthesise. In both cases, we were able to attach parts together easily using PCR and Gibson assembly.
The products of Gibson Assembly reactions were then transformed into E. coli (NEBTurbo) competent cells (made in-house). These colonies were selected on antibiotic-containing LB agar plates before colony PCR and validation by Sanger sequencing (Source Bioscience).
One of the main components of our experimental work was focused around CD27L, an endolysin from ɸCD27 bacteriophage that targeted C. difficile. CD27L is a protein with two domains: an N-terminal N-acetylmuramoyl-L-alanine amidase homologue and a C-terminal domain. It has been argued that the N-terminal domain has the same specificity as the full length CD27L and improved catalytic activity.2
We were interested in testing different versions of the endolysin (one from Team Dundee 2012 as well as a truncated and full-length endolysin) in different conditions, to see how it behaves in different environments. Thus, to carry out those experiments, we expressed both the CD27L and CD27L1-179 endolysins, allowing us to compare them and test how they behave.
Figure 2. Constructs of CD27L Parts:
For our purification strategy, we expressed the endolysins in E. coli and purified it via immobilized metal ion affinity chromatography (IMAC) to obtain a partially purified protein solution. Then, after confirming by SDS-PAGE that we had the correct product, we dialysed our protein into phosphate buffered saline (PBS). With such a purification strategy, we aimed to obtain a pure enough sample of active endolysin for killing assays.
To express our endolysin, we fused our endolysin with a N-terminal 6x-His tag and a SpyTag3, which enables the utilisation of two different affinity chromatography (AC) methods, as well as tandem affinity purification (TAP).
The constructed part was cloned into an IPTG-inducible, T7 expression system-based pET28a vector. For our host organism, we used the BL21(DE3)-RIPL expression strain, which is optimized for the T7 expression system and rare codons. We grew our cells in LB at 37°C and then induced the cultures in mid-log phase with IPTG at 180RPM 21°C;. Following induction overnight, the cells were harvested using centrifugation and lysed by sonication. Purification was then carried out as mentioned above.
Our choice of buffer was based on previous kill assays in literature, which used PBS as the main buffer for the reaction2. Specifically, in cases where our yield was low (i.e. CD27L1-179 construct), we used spin concentration to achieve total protein concentration suitable for killing assays.
During purification, we encountered several issues with both the CD27L and CD27L1-179 proteins. The latter was expressed at insufficiently high concentrations for assaying, so spin concentration was used to achieve high enough concentrations.
Additionally, our progress with CD27L was also hindered by the precipitation of our samples during dialysis. Specifically, our CD27L endolysin precipitated in substantial quantities (protein concentration decreasing from 5mg/ml to 0.3mg/ml) in three batches of our five.
Our initial hypothesis was that this occurred due to the lack of reducing conditions. This hypothesis was based on the structural model of CD27L, where it is shown there are several surface cysteine residues. However, adding beta-mercaptoethanol to all steps of purification, including dialysis, did not resolve this issue. Thus, to fully address this question, further investigation is required.
Tandem Affinity Purification (TAP) was also planned to obtain higher purity samples. However, we decided that our one step affinity chromatography yielded sufficient purity for our particular CD27L applications.
To test the CD27L endolysin, we had to create an assay demonstrating whether it is able to function as it was shown in the literature2,4. From such preliminary research, we discovered that CD27L, besides being active against Clostiridoides difficile, is also able to cleave amide bonds in the cell wall of other bacteria with the same peptidoglycan type, A1γ. Thus, we used Bacillus subtilis, a Category 1 Gram-positive bacterium2 as our surrogate target due to safety concerns with the Category 2 C. difficile.
In order to assay whether B. subtilis is lysed by our endolysin, we measured absorbance at 600nm (OD600) throughout an 8 hour time period at every 15 minutes. This way, we were able to monitor how the B. subtilis culture is affected by CD27L.
Notably, our kill assay differs from the one described by Mayer et al. (2008)4. Their protocol involved washing and concentrating the cells to OD600 = 2.0 in Phosphate Buffered Saline (PBS), followed by mixing with CD27L and measuring the rate of decrease in OD600.
However, our protocol involved growing up a B. subtilis culture until they reached mid-log phase (OD600 ≈ 0.6), then diluting them in Lysogenic Broth (LB), adding our solution (CD27L or Lysozyme or PBS) and then measuring the growth over time by monitoring the change in OD600. The measurements were made automatically by a plate reader, BMG FLUOstar Omega and the cells were grown in a 96 well plate from Greiner (CellStar® 96 well plate F-type).
To explore the capabilities of the endolysin, we carried out killing assays under different conditions. First, we tested the concentration dependence of our endolysin activity. Second, we tested the specificity of our endolysin by assaying CD27L against both E. coli and L. reuteri.
Given that our CD27L construct has a SpyTag, we were also able to attempt protein oligomerisation to see greater activity from the oligomer. The SpyTag/SpyCatcher system allows for irreversible protein conjugation through the formation of an isopeptide bond. Oligomerisation can be achieved using previously characterised SpyCatcher-o-Hept. This contains SpyCatcher/Coiled-Coil monomers that forms heptamers in solution5. Although we attempted oligomerisation, our data were ultimately inconclusive.
Additionally, we performed scanning electron microscopy on our B. subtilis samples. Such a technique allowed us to visually confirm changes in B. subtilis cell wall morphology upon application of endolysin. This supports the hypothesis that the endolysin acts to degrade the peptidoglycan cell wall, ultimately causing lysis.2
Figure 3. SEM Images of B. subtilis Post-Treatment with CD27L Endolysin
Our part improvement revolved around the comparison between our full length CD27L endolysin construct against the 2012 iGEM Dundee part BBa_K895005, as further detailed on the Part Improvement page.
Experimentally, we cloned the construct as in Figure 1 into the pET28a vector (IPTG-inducible, T7 expression system). The particular construct included an HA-tag, allowing for affinity labelling for Western Blot. Assembled constructs were transformed into BL21(DE3)-RIPL cells. Upon induction however, both SDS-PAGE gels and Western blots gave negative results, hence a lack of expression. Our solution was thus to remove the VgrG Type VI secretion protein on the original part, and repeat our efforts to express this part.
Figure 4. Dundee 2012 iGEM Part BBa_K895005 Construct
Our ProQuorum system relies on the detection of the C. difficile-specific quorum sensing signalling molecule known as AIP (autoinducer peptide). This molecule is hypothesized to be a small cyclic peptide that is synthesized from AgrD (precursor) by AgrB (a transmembrane protein) 6.
Figure 5. Transmembrane Prediction for AgrB Protein via TMHMM 2.07
We attempted to create a recombinant construct of AgrBD to produce recombinant AIP. This would have allowed us to test our AgrAC detection system in L. reuteri. A similar approach has been carried out previously with Staphylococcus aureus8.
Initially, our construct was designed in the arabinose-inducible pBAD vector. However, due to difficulties with induction, we switched to the IPTG-inducible pET vector, as seen below.
Figure 6. AgrBD Construct
We also explored the novel affinity chromatography method of Spy&Go purification. This method uses a fusion tag, called SpyTag developed by Zakeri et al. (2012)3 which binds to its stationary phase called SpyDock via noncovalent interactions. The SpyTag fused protein, after binding, can be eluted conveniently with 2.5 M imidazole (a relatively cheap compound). Moreover, the purification itself is claimed as a more sensitive alternative to His-tag Ni-NTA purification method5.
To test this purification, we used mClover3, a bright and photostable green fluorescent protein which has not previously been registered in the Part Registry, fused with 6xHis and SpyTag fusion tags9.
The protein was cloned into an IPTG inducible pET28a vector containing a T7 expression system. The assembled vector was transformed into BL21(DE3)-RIPL expression strains. We grew our cells in LB at 37°C; and induced the cultures in mid-log phase with IPTG at 180RPM 21°C;. The cells were harvested through centrifugation and lysed using sonication.
Since both Ni-NTA and Spy&Go purification methods require different buffer conditions, we halved each culture before sonication and resuspended them in the optimal buffer for lysis. After obtaining a clarified lysate through centrifugation, the lysate was loaded onto either a SpyDock or Ni-NTA column. The recommended protocols were used for both purification steps and 0.5mL elution fractions were collected. The purification was then checked by SDS-PAGE.
Subsequently, we would ideally have a convenient method of assessing the purity of our samples. However, we were not able to obtain samples of sufficient purity to perform assay based quantification. Instead, we utilized densitometric measurements in SDS-PAGE gels after Coomassie Blue staining using BioRad’s Chemidoc XRS and Image Lab (6.0.1) software to measure Lane% (which we refer to as purity), as shown in Figure 2 below.
Figure 6. Comparison of Spy&Go Purification vs. Ni-NTA purification by Densitometry
