Team:TAU Israel/Results
Introduction
The objective of our project was to find new ways to fight antibiotic resistant bacteria, as described in the project's description. We were inspired by previous studies and chose to address this issue by re-targeting R2-type pyocins[1]. In WT Pseudomonas aeruginosa PAO1, the R2-type pyocin gene cluster has been shown to be under the regulation of the bacteria's SOS response. The SOS response triggers the activation of RecA, which in turn degrades the prtN gene repressor called prtR. Following prtR's degradation, the downstream-encoded prtN gene is expressed. PrtN serves as an activator of the R2-type pyocin gene cluster expression[2].
Our System
The system regulation
In order to achieve expression of multiple re-targeted pyocins, we set out to create two regulations for the gene cluster expression, based on two different sequences up-stream to the partial cluster first structural gene.
The first regulation (long, Fig. D.3) is similar to the WT regulation that PAO1 exhibits. RecA+ E. coli were transformed with the gene cluster including prtR and prtN, and the other essential genes for the cluster assembly and release, encompassing the genes encoding prf5-prf24, which were shown to be sufficient for pyocin assembly and activity in a previous study[3].
The second regulation (short, Fig. D.4) was based on our idea to separate prtN, the gene that encodes for the cluster activator, from the negative regulation of prtR, and therefore separate it from the rest of the gene cluster. We decided to insert the prtN gene into a Marionette plasmid, under the induction of vanillic acid[4], whereas the gene cluster was inserted into another vector, relying on the induction of prtN.
Promoter detection
After omitting prtR and prtN from the cluster, we had to decide where the cluster should start. To that end, we used bioinformatic tools to compare and identify the consensus promoter of the cluster. This work is detailed in the bioinformatics work page. Based on our findings, we decided to start our cluster 300bp up-stream of the first structural gene prf5. This 300bp sequence contains the promoter that we identified, and perhaps includes additional sequences that are important for the cluster expression as well.
Pyocin re-targeting
The two systems that we constructed share a deletion mutation in the prf15. prf15 encodes for the tail fiber protein, hence it is responsible for targeting a specific bacterial target, which is another strain from the same species. This mutation was previously shown to be effective in order to successfully re-target R2-type pyocins towards novel bacteria[3][5][6]. Therefore, by creating this mutation and introducing another plasmid expressing an alternative tail fiber protein, we aimed to target new bacteria. The different tail fibers were induced by IPTG, while each tail fiber was cloned separately inside the vector. The tail fibers we chose were shown to effectively kill their target bacteria[3][5][6]. In addition, based on our bioinformatic work, we identified another tail fiber candidate that originates in FELS2 bacteriophage, that targets Salmonella LT2.
Figure A.1: A scheme showing how we isolated each part of the cluster into a different plasmid in order to express re-targeted pyocins in E.coli.
Figure A.2: The pyocin expression signaling pathway, which starts with the activation of RecA and ends with the expression and release of assembled pyocins.
PrtN plasmid
About the plasmid
prtN originates from Pseudomonas aeruginosa , and is used as an activator in our system[1][2]. prtN (345 bp) was inserted into E. coli[4], inside Marionette (pajm.1642) plasmid (3.2kbp) - a medium copy number plasmid [Fig B.1.]. This plasmid is part of the Marionette Sensor Collection, which enables modular control of up to 12 genes, by placing each one under the control of an inducible promoter. The backbone plasmid contained kanamycin resistance and the expression of a desired protein can be induced by adding vanillic acid. This plasmid was chosen since the expression from its promoter was shown to be less “leaky” than other plasmids offered in the Marionette system[4].
Gibson Assembly Cloning
We ordered prtN as a G-block and amplified the backbone from the Marionette pAJM.1642 plasmid using PCR [Fig B.2]. We purified the PCR reaction and inserted prtN into the backbone using the Gibson assembly method. The resulting plasmid was transformed into E. coli DH10β, and the transformants were incubated overnight under selective conditions. Next, we performed a colony PCR on 3 resulting colonies to confirm that they contain the desired constructed plasmid. Finally,we purified the plasmids from the E. coli colonies using a miniprep kit and sent them for sequencing to confirm.
Fig B.1. prtN plasmid in our system.
Fig B.2. Gel Electrophoresis for backbone plasmid. Backbone size is 3.2kbp
Tail plasmids
Four different tail plasmids were constructed (Fig. C.1). Each plasmid codes for a unique tail fiber protein under an inducible regulation (we chose to use IPTG as the inducer). All tail plasmids share the same selective marker (ampR) and high copy origin of replication.
Prf15 tail plasmid codes for the tail fiber protein (prf15) and its chaperone (prf16) from Pseudomonas aeruginosa PAO1. The coded tail plasmid protein allows pyocin particles to target rival Pseudomonas strains.
prf15-p2 fusion tail plasmid codes for the N-terminal part (164 A.A) of the tail fiber protein (prf15) from Pseudomonas aeruginosa PAO1 fused with the C-terminal part (512 A.A) of the P2 bacteriophage tail fiber protein alongside its chaperone. Pyocins with this tail fiber will target a wide range of E. coli strains.
prf15-V10 fusion tail plasmid codes for the N-terminal part (164 A.A) of the tail fiber protein (Prf15) from Pseudomonas aeruginosa PAO1 fused with the C-terminal part (658 A.A) of the V10 bacteriophage tail fiber protein. Pyocins with this tail will target strains that present the antigen O157 (such as E. coli EDL933 (O157:H7)).
Fels2 tail plasmid is the result of our unique tail finding software! (see software page for more info about our bioinformatic work). We have used our tail finding algorithm and discovered a tail fiber gene from the bacteriophage Fels-2, that targets Salmonella Typhimurium. We predicted that the Fels2 tail protein can be incorporated into the pyocin assembly. Following this finding, we constructed this plasmid to re-target pyocins against Salmonella Typhimurium. This plasmid includes a tail fiber and tail chaperone genes.
The unique insert in each plasmid was ordered as a G-block (prf15-P2 fusion, prf15-V10 fusion and Fels2 plasmids) or amplified from the genome of Pseudomonas aeruginosa PAO1 (prf15 tail plasmid, see PCR results in Fig. C.2). As can be seen in Fig. C.3 (protein gel), there is an expression of prf15-p2 fusion protein and its chaperone (although the chaperone is shorter than what we expected). Prf15 and prf16 are also expressed. The promoter seems to be very leaky - and yet induction with IPTG results in higher expression.
Fig. C.1 – Tail plasmids' map. All tail plasmids share the same inducible regulation, selective marker (ampR) and high copy origin of replication.
Fig. C. 2 – PCR results: amplification of prf15+prf16 from the genome of Pseudomonas aeruginosa PAO1. From left to right: two ladders and 4 PCR reactions at different annealing temperatures.
Fig. C.3 – protein gel for the different tail proteins and prtN. i = induced, m = mock (not induced).
Cluster plasmid
About the plasmid
This plasmid contains a partial R-type pyocin gene cluster (prf5–prf24 genes) from Pseudomonas aeruginosa PAO1, with a partial deletion of the tail gene (prf15). An in-frame deletion of codons 11 to 301 of prf15 was done according to a report on replacing pyocin tail fiber proteins[3]. The partial cluster in our plasmid contains the promoter sequence (300 bp up-stream of prf5), the structural genes, the tail fiber’s chaperone (prf16) and lysis regulating genes (prf9 & prf24). Expression of this cluster is controlled by the original activator, prtN, whose expression is controlled by either vanillic acid-mediated induction or by prtR degradation induced by Mitomycin C.
Due to difficulties that arose during the PCR stage, we have decided to construct this clone in two different methods - Gibson Assembly and Golden Gate.
Gibson assembly cloning
In this method, we divided the original pyocin cluster into two parts: the first part consists of genes prf5-prf15 and the second part consists of genes prf16-prf24 [Fig.D.1]. We chose a low copy number plasmid with psc101 origin of replication as our backbone plasmid, which contains chloramphenicol resistance as a selection marker.
In the first stage, we amplified the two parts from P. aeruginosa PAO1 and the backbone through PCR reaction [Fig. D.2]. Afterward, we inserted the two parts into the backbone plasmid using the Gibson Assembly method and transformed it into E.coli DH10β. We then sequenced the plasmid, but due to technical problems the results of the sequencing were unreliable, and due to lack of time we did not repeat this stage.
Fig. D.1: Cluster plasmid map
Fig. D.2: Cluster PCR gel electrophoresis results.
1 - Second part of the cluster, 8.9 Kb
2 - First part of the cluster, 5.07 Kb
Golden Gate cloning
For the Golden Gate cloning method, we decided to order synthetic parts of the cluster instead of trying to amplify it by PCR. We needed to split the cluster into 6 different parts that are up to 3.5 kb each. The first part has two versions - long and short (fig. D.3 and D.4). The two regulation we constructed rely on these two different parts as detailed in the results introduction. The rest of the 5 parts contain the structural genes and the lysis genes that are necessary for the pyocin assembly and they were used for both regulation options.
First, in order to clone our cluster into the desirable backbone (low copy number, Chloramphenicol resistance), we needed to insert a lacZ gene into this plasmid. To that end, we used Gibson assembly cloning protocol (Fig. D.5) and then seeded the transformed bacteria on blue-white screening plates (Fig. D.6). Then, we used the blue colonies for the backbone purification. The cluster parts that we received as plasmids from Genescript were transformed separately in DH10β. In the next step, we mixed the parts' plasmids with our lacZ backbone in a Golden Gate assembly reaction, according to the first regulation and the second regulation described in the introduction. The resulting solutions were used in transformation for blue-white screening (Fig. D.7). We picked the white colonies and purified them for following transformations with the tails' plasmids and prtN plasmid (for the second regulation). BL21 E. coli were transformed with the first version cluster plasmid and DH10β were transformed with the second version cluster plasmid.
Fig D.3: The first part of the first version of the cluster. It includes prtN, prtR , their assumed terminator[2] (gray) and the assumed cluster promoter as identified by us (light red). The restriction sites are indicated in light blue on both sides of the sequence.
Fig D.4: The first part of the second version of the cluster. It includes prtR with double stop mutations (red) and the assumed cluster promoter as identified by us (light red). The mutation was made in order to allow the presence of a much longer sequence up-stream to prf5 together with a dysfunctional PrtR. The restriction sites are indicated in light blue on both sides of the sequence.
Fig D.5: PCR products of lacZ plasmid fragments for Gibson assembly. As expected, the lacZ insert is between 500-600 bp and the linear backbone fragment is 4.5 kb.
Figure D.6: DH10β blue colonies after transformation with the lacZ Gibson reaction product.
Figure D.7: DH20β white colonies after transformation with GG reaction product. In this picture, only the second version of the cluster appears, but white colonies were detected for the first version as well (not shown).
Promoter Binding Detection
We used bioinformatic tools in order to find the location of the pyocin cluster’s promoter. We retrieved the genomes of hundreds of Pseudomonas strands and created their consensus sequence by applying multiple sequence alignment (using MAFFT) on the cluster area. Inside the consensus sequence, we looked for conserved sites with the RNA Polymerase motif and we were able to identify such area about 94 nucleotides before the cluster.
Plaque assay
We have conducted three plaque assays to test our engineered E. coli.
In the first plaque assay, we wanted to assess natural pyocin activity. We used Mitomycin C that induces SOS response to trigger Pseudomonas aeruginosa PAO1 to produce pyocins and lyse. Then, we spotted the clear PAO1 lysate on R2-type pyocin-sensitive (S13 and PAK) and non-sensitive (PAO1 and PA14) Pseudomonas strains. The following day, we checked and discovered plaques caused by pyocins in the lysates we produced! Importantly, there were no plaques in the negative controls (non-sensitive target strains). Fig. E.1.
Fig. E.1 - Plaques caused by R2-type pyocins on Pseudomonas PAK plate. 3 samples caused plaques – Pseudomonas PAO1 treated with Mitomycin C, Pseudomonas PAO1 treated with Mitomycin C (concentrated lysate) and Pseudomonas PAO1 without Mitomycin treatment. LB and E. coli (negative controls) treated with Mitomycin C did not cause plaques.
In the second plaque assay, we tested the engineered E. coli with the plasmids we constructed using Gibson reactions. We cloned bacteria with tail plasmid, activator plasmid and cluster plasmid (different clones for different tails). We also cloned negative controls (bacteria with an "empty" activator/cluster plasmid). Each strain was grown and treated with IPTG and vanillic acid to activate pyocin assembly and lysis. PAO1 was grown and activated with Mitomycin C as a positive control. Unfortunately, when we looked at the bacterial suspensions the next day, they were not clear, suggesting that the cells were not lysed (except for the positive control).
For the targets E. coli and Salmonella, plaques were found all over the plates, also in locations that we spotted with supernatants from negative control strains. We hypothesized that there were some antibiotics left in the supernatants, and that the antibiotics are responsible for the plaques we saw. For the target Pseudomonas S13, which is resistant to the antibiotics we used to grow the engineered E. coli, plaques emerged only in the areas treated with supernatant from the positive control strain.
In the third plaque assay, we tested the engineered E. coli with the plasmids we constructed using Gibson reactions (again) and the engineered E. coli with the plasmids we constructed using Golden Gate reactions (for the first time). Each strain was grown and induced by the relevant inducers to activate pyocins assembly and lysis. We observed again that the cells did not look like they were lysed after induction (except for the positive control). We have carried out the experiment and performed the plaque assay. The results can be seen in the following pictures (Fig. E.2-4): All supernatants (including the negative controls) caused plaques in the Salmonella plate. This target is probably extremely sensitive to the remaining antibiotics in the supernatants we used. For the E. coli plate, there was only one supernatant that caused a plaque. This supernatant was made from bacteria that did not grow over night at the beginning of the experiment. We think that this supernatant had a higher amount of remaining antibiotics because there were no bacteria in the sample to consume it, and that this is the reason for the plaque we see. For the Pseudomonas S13 plate, only the supernatant from the positive control caused a plaque.
Overall, we did not observe activity of our engineered pyocins. As the cells were not lysed, future attempts should try and test a few options that could fix this problem: adjusting inducers concentration, lysing the cells mechanically as part of the protocol or using lysis genes in the system. In addition, future attempts can try and clean the lysate from remaining antibiotics or produce antibiotic-resistant targets.
Fig. E.2 - Salmonella Typhimurium plate
Fig. E.3 - E. coli plate
Fig. E.4 - Pseudomonas S13 plate
Conclusion
As demonstrated and mentioned before, the pyocin system has great potential, but a significant part of it still has to be explored. Pyocins are characterized by high specificity, they carry no foreign genetic cargo and their target range can be manipulated. As part of our work, we aimed to re-engineer the natural pyocin system (similar to what has been earlier done by Dean Scholl) by separating it into three parts: Cluster, Tail and Regulatory complexes.
During this work, we encountered difficulties mainly in the field of plasmid design and construction due the complexity of the cluster. Eventually we created all three plasmids, sequenced them and then transformed all three into E. coli. However, as can be seen in the results above, we have yet to overcome the challenge of properly expressing all necessary cluster proteins and regulators together. We conclude that additional work is required to allow expression of all system components; yet we are confident that the task is feasible and will ultimately lead to the construction of an effective and controllable anti-bacterial tool!
Future plans
As mentioned above, there is further work that still has to be done, primarily in achieving proper protein expression, but also in more advanced fields such as drug delivery and protein purification methods. As can also be seen in our Human Practices section, we held a meeting with the head of the Israeli center for antibiotic resistant bacteria. During this meeting, we received important insights regarding the possible difficulties in creating an on-site drug factory. One such difficulty would be the immunogenic response to the presence of the foreign bacteria or protein, another possible challenge would be the survival rate of the drug factory itself. Thus, apart from improving the protein expression profile of the system, we suggest to focus on purification methods for the pyocins, allowing for low complexity, low risk delivery of the pyocins to the target site
Improving the modularity and regulation of the system
An important phase in the development of the system would be to make it more modular and tightly regulated. In some of the experiments that were carried out, we encountered leakiness of the promoters or insufficient expression on others. Another possible improvement we would consider is to completely separate the pyocin cluster from it’s original regulatory system, i.e. placing it under the regulation of a highly characterized promoter from the repository of standard biological parts.
Pyocins purification and quantification
In a consultation we’ve held with Mr. Dean Scholl, we inquired about methods for purification of the pyocins out of the cell lysate. We have also found out that according to his work, it might be useful to grow bigger stocks of the pyocins prior to the plaque assay to achieve higher concentration and better results.
Another aspect is a higher quality of quantification, this might be achieved by creating multiple dilutions of purified pyocin lysate and then incubating it with a known quantity of target bacteria. The contents of the microwells with the target bacteria will be then diluted and spotted on agar plates and the surviving bacteria will be counted to estimate the survival rate and the activity of the pyocins.