Presented by Team BOKU-Vienna 2020
Oona Jung¹, Katja Starnberger¹, Lukas Pucher¹, Benedikt Haslinger¹, Dominik Wiesinger¹, Katharina Ostertag¹, Lukas Pranter¹, Samuel Oberreiter¹, Rajmund Kasbauer¹, Markus Strasser¹, Simon Wutzl¹, Harriet Schober¹, Styliani Karagiorgou¹, Martin Altvater², and Diethard Mattanovich²
¹iGEM Student Team Member, ²iGEM Team Primary PI
Abstract
Our vision is to alleviate bacterial infections and their systemic consequences by not only eliminating hostile bacteria but also, in a second step, capturing their toxic components released during bacterial lysis. Our goal is to recombinantly engineer a T7 bacteriophage that by infection forces its target bacterium to produce gelsolin, a protein able to bind the endotoxin LPS. Firstly, we designed our experiments in silico to contribute our parts to the iGEM registry. We then cloned the plasmids containing the recombination system necessary for engineering our phage. The genes to be inserted into the phage genome were amplified. Additionally, we designed a safety measure to inhibit the engineered phage’s ability to reproduce autonomously. To support our experimental findings we modelled the interaction between T7 bacteriophages and E.coli. Furthermore, to gain more insight into the biology of the T7 bacteriophage, we collaborated with team TU Delft to characterize the wildtype.
Team:BOKU-Vienna/Poster
Poster: BOKU-Vienna
PHANGEL - Taking Phage Therapy Ahead
Project goals
Our overall goal was to develop a therapy supporting or alternative to common antibiotic therapy to improve the prognosis of sepsis patients and accelerate their recovery. To achieve this main goal, we divided it into smaller steps:
1. We aimed to create a genetically engineered bacteriophage able to induce the production of Human Plasma Gelsolin, the peptide containing gelsolin’s LPS binding site GSN 160-169 or the antimicrobial peptide 19-2.5.
2. We aimed to develop a safety mechanism that was simple and transferrable, yet efficient as to be able to control the given dosage and not harm the patients.
3. We aimed to make the recombination system accessible in the iGEM registry for future teams to use.
1. We aimed to create a genetically engineered bacteriophage able to induce the production of Human Plasma Gelsolin, the peptide containing gelsolin’s LPS binding site GSN 160-169 or the antimicrobial peptide 19-2.5.
2. We aimed to develop a safety mechanism that was simple and transferrable, yet efficient as to be able to control the given dosage and not harm the patients.
3. We aimed to make the recombination system accessible in the iGEM registry for future teams to use.
Inspiration
Estimations suggest that more than 11 million people die of sepsis every year. That is one in five deaths around the world and makes sepsis the most prevalent cause of death, even more deadly than cancer.1 Sepsis is caused by pathogenic infections, during which endotoxins are released from the bacterial membrane that cause a dysregulated systemic immune reaction in which the immune system attacks endogenous tissues.2
This topic is especially urgent in times like ours when an increasing number of pathogens cannot be combated with well-known strategies such as antibiotics anymore. Studies show that it can take less than a year for a resistance against a newly discovered antibiotic to be evolved and spread.3 Thus, alternative therapies to common antibiotic treatment are desperately needed.
One very promising branch of research in the search for new ways to combat bacterial infections are bacteriophages (phages). In the last few decades, these viruses came into scientific focus due to their ability to kill bacteria. However, phage therapy also bears risks and it is important to overcome them in order to help patients.
1https://www.bbc.com/news/health-51138859#:~:text=One%20in%20five%20deaths%20around,than%20are%20killed%20by%20cancer. (last seen on: 26.10.2020)
2https://flexikon.doccheck.com/de/Sepsis (last seen on: 26.10.2020)
3https://www.cdc.gov/drugresistance/about.html (last seen on: 26.10.2020)
1https://www.bbc.com/news/health-51138859#:~:text=One%20in%20five%20deaths%20around,than%20are%20killed%20by%20cancer. (last seen on: 26.10.2020)
2https://flexikon.doccheck.com/de/Sepsis (last seen on: 26.10.2020)
3https://www.cdc.gov/drugresistance/about.html (last seen on: 26.10.2020)
Problem
Phages alone do not tackle the problem of sepsis. The opposite might even be true. When a phage kills a bacterium in the so called “lytic cycle”, the membrane of the bacterium is destroyed, releasing all its endotoxic components. In gram-negative bacteria such as E.coli up to 75% of the outer cell membrane can consist of lipopolysaccharides, short LPS. The endotoxin LPS is heat- and pH stable and often involved in the origin of a sepsis as they elicit strong immune responses and weaken the immune system.4
Simplified, this immune response is mediated in the following way: LPS is opsonized by the LPS-binding protein (LBP). This complex is recognised by glycoprotein CD14 which, together with Toll-like receptor 4, forms the LPS receptor on macrophages. If the LPS-LBP complex binds to the LPS receptor, a signal cascade is induced that leads to the release of proinflammatory factors such as cytokines, TNF‐α, IL‐1β and IL‐6 which causes the systemic inflammation.5
This is a potentially dangerous side effect of bacterial degradation and already a matter of research among phage scientists: several studies have been conducted to impair or inhibit the lysis of host bacteria in order to keep the endotoxin contained. In several different studies genetically engineered phages were created to kill the bacteria not through lysis but through degradation of their genome keeping the membrane mostly intact.6 However, we know of no approach like PHANGEL.
4https://www2.biomin.net/at/artikel/the-hidden-dangers-of-lipopolysaccharides/ (last seen on: 26.10.2020)
5Cohen, J. The immunopathogenesis of sepsis. Nature 420, 885–891 (2002). doi: 10.1038/nature01326)
6Pires, D. P., Cleto, S., Sillankorva, S., Azeredo, J., & Lu, T. K. (2016). Genetically Engineered Phages: a Review of Advances over the Last Decade. Microbiology and molecular biology reviews : MMBR, 80(3), 523–543. doi: 10.1128/MMBR.00069-15
Simplified, this immune response is mediated in the following way: LPS is opsonized by the LPS-binding protein (LBP). This complex is recognised by glycoprotein CD14 which, together with Toll-like receptor 4, forms the LPS receptor on macrophages. If the LPS-LBP complex binds to the LPS receptor, a signal cascade is induced that leads to the release of proinflammatory factors such as cytokines, TNF‐α, IL‐1β and IL‐6 which causes the systemic inflammation.5
This is a potentially dangerous side effect of bacterial degradation and already a matter of research among phage scientists: several studies have been conducted to impair or inhibit the lysis of host bacteria in order to keep the endotoxin contained. In several different studies genetically engineered phages were created to kill the bacteria not through lysis but through degradation of their genome keeping the membrane mostly intact.6 However, we know of no approach like PHANGEL.
4https://www2.biomin.net/at/artikel/the-hidden-dangers-of-lipopolysaccharides/ (last seen on: 26.10.2020)
5Cohen, J. The immunopathogenesis of sepsis. Nature 420, 885–891 (2002). doi: 10.1038/nature01326)
6Pires, D. P., Cleto, S., Sillankorva, S., Azeredo, J., & Lu, T. K. (2016). Genetically Engineered Phages: a Review of Advances over the Last Decade. Microbiology and molecular biology reviews : MMBR, 80(3), 523–543. doi: 10.1128/MMBR.00069-15
Idea
Our idea PHANGEL is a genetically modified bacteriophage that not only presents an attractive alternative to traditional antibiotic therapy against bacterial infections but also tackles the adverse effects arising from bacterial lysis.
We aimed to engineer a bacteriophage that would not only kill the harmful host bacterium but would force it to express a LPS binding substrate in order to prevent the uncontrolled release of LPS into the system during lysis. As LPS binding and inactivating substrates we chose human plasma gelsolin, a peptide of the LPS-binding region GSN 160-169 or the antimicrobial peptide 19-2.5.7,8,9 We planned to replace the gene for the Major Capsid protein (necessary for phage assembly) in the T7 phage genome with these proteins and peptides which at the same time disables the uncontrolled replication of the engineered phages.
7Schuerholz, T., Doemming, S., Hornef, M., Martin, L., Simon, T. P., Heinbockel, L., Brandenburg, K., & Marx, G. (2013). The anti-inflammatory effect of the synthetic antimicrobial peptide 19-2.5 in a murine sepsis model: a prospective randomized study. Critical care (London, England), 17(1), R3. doi: 10.1186/cc11920
8Cheng Y, Hu X, Liu C, Chen M, Wang J, Wang M, Gao F, Han J, Zhang C, Sun D, Min R: Gelsolin Inhibits the Inflammatory Process Induced by LPS. Cell Physiol Biochem 2017;41:205-212. doi: 10.1159/000456043
9Bucki R, Georges PC, Espinassous Q, Funaki M, Pastore JJ, Chaby R, Janmey PA. Inactivation of endotoxin by human plasma gelsolin. Biochemistry. 2005 Jul 19;44(28):9590-7. doi: 10.1021/bi0503504. PMID: 16008344.
7Schuerholz, T., Doemming, S., Hornef, M., Martin, L., Simon, T. P., Heinbockel, L., Brandenburg, K., & Marx, G. (2013). The anti-inflammatory effect of the synthetic antimicrobial peptide 19-2.5 in a murine sepsis model: a prospective randomized study. Critical care (London, England), 17(1), R3. doi: 10.1186/cc11920
8Cheng Y, Hu X, Liu C, Chen M, Wang J, Wang M, Gao F, Han J, Zhang C, Sun D, Min R: Gelsolin Inhibits the Inflammatory Process Induced by LPS. Cell Physiol Biochem 2017;41:205-212. doi: 10.1159/000456043
9Bucki R, Georges PC, Espinassous Q, Funaki M, Pastore JJ, Chaby R, Janmey PA. Inactivation of endotoxin by human plasma gelsolin. Biochemistry. 2005 Jul 19;44(28):9590-7. doi: 10.1021/bi0503504. PMID: 16008344.
Engineering
One of the best described methods to engineer phage genomes described is the BRED system.10 BRED stands for Bacteriophage Recombineering of Electroporated DNA and was first used to modify difficult to engineer mycophages.11 The principle is the following: the purified linear phage DNA and the double-stranded linear DNA to insert/delete/replace in the phage (both are called the recombineering substrates) are co-electroporated into electrocompetent bacterial cells which carry a recombination plasmid, i.e., a plasmid which expresses proteins that promote a high degree of homologous recombination. For our project, we decided on the well studied Lambda RED system 12, consisting of the exo, beta and gam. Exo is the lambda exonuclease that binds and degrades dsDNA non-sequence-specific from 5’ to 3’ , reforming the 5’-end into a 3’overhang 13,14 and leaving ssDNA and promoting the annealing or loading of Beta. Beta binds to the single-stranded version made by Exo and facilitates the recombination of the homologous sequences. Gam protects the ssDNA produced by Exo from endogen E.coli nucleases.13,15 This means that the insert needs to have at least 80bp of homologous sequences to the desired place of insertion in the T7 genome. These homologous sequences are called recombination sites.
To engineer our T7 bacteriophage, we designed the Lambda RED Recombination system on a plasmid (BBa_K3514004). The sequences of LPS binding proteins and peptides we wanted to insert were modified to specifically replace the Major Capsid protein of the T7 phage. The special recombination sites were added to the gene constructs to achieve this. These sequences were not added to the registry as they are only relevant for our specific project.
10Marinelli LJ, Hatfull GF, Piuri M. 2012. Recombineering: a powerful tool for modification of bacteriophage genomes. Bacteriophage 2:5–14. doi: 10.4161/bact.18778.
11Marinelli LJ, Piuri M, Swigonová Z, et al. BRED: a simple and powerful tool for constructing mutant and recombinant bacteriophage genomes. PLoS One. 2008;3(12):e3957. doi: 10.1371/journal.pone.0003957
12Christopher R. T. Hillyar ;Genetic recombination in bacteriophage lambda; 2012; Bioscience Horizons: The International Journal of Student Research, Volume 5, 2012, hzs001, doi: 10.1093/biohorizons/hzs001
13Lambda Red Recombineering in Escherichia coli Occurs Through a Fully Single-Stranded Intermediate J. A. Mosberg,*†,1,2 M. J. Lajoie,*†,1,2 and G. M. Church* Genetics. 2010 Nov; 186(3): 791–799.
14Szczepanska, A. K., 2009. Bacteriophage-encoded functions engaged in initiation of homologous recombination events. Crit. Rev. Microbiol. 35 197–220.
15Sawitzke, J. A., L. C. Thomason, N. Costantino, M. Bubunenko, S. Datta et al., 2007. Recombineering: in vivo genetic engineering in E. coli, S. enterica, and beyond. Methods Enzymol. 421 171–199.
10Marinelli LJ, Hatfull GF, Piuri M. 2012. Recombineering: a powerful tool for modification of bacteriophage genomes. Bacteriophage 2:5–14. doi: 10.4161/bact.18778.
11Marinelli LJ, Piuri M, Swigonová Z, et al. BRED: a simple and powerful tool for constructing mutant and recombinant bacteriophage genomes. PLoS One. 2008;3(12):e3957. doi: 10.1371/journal.pone.0003957
12Christopher R. T. Hillyar ;Genetic recombination in bacteriophage lambda; 2012; Bioscience Horizons: The International Journal of Student Research, Volume 5, 2012, hzs001, doi: 10.1093/biohorizons/hzs001
13Lambda Red Recombineering in Escherichia coli Occurs Through a Fully Single-Stranded Intermediate J. A. Mosberg,*†,1,2 M. J. Lajoie,*†,1,2 and G. M. Church* Genetics. 2010 Nov; 186(3): 791–799.
14Szczepanska, A. K., 2009. Bacteriophage-encoded functions engaged in initiation of homologous recombination events. Crit. Rev. Microbiol. 35 197–220.
15Sawitzke, J. A., L. C. Thomason, N. Costantino, M. Bubunenko, S. Datta et al., 2007. Recombineering: in vivo genetic engineering in E. coli, S. enterica, and beyond. Methods Enzymol. 421 171–199.
Safety
The integration of a safety mechanism in our project design is a necessity since we aim for our GMO to be applied in the human body. Our solution is to produce a phage which is not able to self-replicate: We replace the gene coding for the major capsid protein, essential for phage assembly and thus replication, with human plasma gelsolin or one of the peptides.
Without the ability to replicate no viable phages can be produced outside of our production strain. The production strain itself carries the gene for the major capsid protein on a separate plasmid and by this is able to generate viable phages.
This mechanism is also highly improbable to fail because our transgene for gelsolin is unlikely to mutate into the major capsid protein or an equivalent protein within one generation. Beyond that, this safety control can easily be transferred to other kinds of phages and makes a potential admission as a therapy more likely.
Parts
The Lambda RED Recombination Plasmid
To engineer our T7 bacteriophage, we designed the Lambda RED Recombination system on a plasmid (BBa_K3514004). The sequences we wanted to insert were modified to specifically replace the Major Capsid protein of the T7 phage. We added all the parts necessary for the Lambda RED Recombination system to the iGEM registry for future teams to use and combined them to a composite part with existing parts from the registry.
Disclaimer: The promoter we used to create the finished recombination plasmid in the registry is not the one we used for our experiments. The promoter we used is also a variant of the T7 Lac promoter that is very well characterized in our facilities. However, it contains sequences that are not compatible with the iGEM registry. As we did not want to mutate this promoter without being able to characterize its mutations, we decided to use the promoter BBa_K2406020 for our contribution to the iGEM registry. In its function and sequence it is very similar to the promoter we used, it is already characterized in the registry and compatible so other teams can use our plasmid.
Safety Mechanism
To ensure a controlled replication of the engineered phages, the major capsid protein of the phages was cloned into a separate plasmid to be transformed into the production strain. We added all the necessary parts to create this plasmid into the iGEM registry and combined them to a composite part with existing parts from the registry.
Disclaimer: The promoter we used to create the finished safety plasmid in the registry is not the one we used for our experiments. The promoter we used is also a variant of the T7 Lac promoter that is very well characterized in our facilities. However, it contains sequences that are not compatible with the iGEM registry. As we did not want to mutate this promoter without being able to characterize its mutations, we decided to use the promoter BBa_K2406020 for our contribution to the iGEM registry. In its function and sequence it is very similar to the promoter we used, it is already characterized in the registry and compatible so other teams can use our plasmid.
To engineer our T7 bacteriophage, we designed the Lambda RED Recombination system on a plasmid (BBa_K3514004). The sequences we wanted to insert were modified to specifically replace the Major Capsid protein of the T7 phage. We added all the parts necessary for the Lambda RED Recombination system to the iGEM registry for future teams to use and combined them to a composite part with existing parts from the registry.
Disclaimer: The promoter we used to create the finished recombination plasmid in the registry is not the one we used for our experiments. The promoter we used is also a variant of the T7 Lac promoter that is very well characterized in our facilities. However, it contains sequences that are not compatible with the iGEM registry. As we did not want to mutate this promoter without being able to characterize its mutations, we decided to use the promoter BBa_K2406020 for our contribution to the iGEM registry. In its function and sequence it is very similar to the promoter we used, it is already characterized in the registry and compatible so other teams can use our plasmid.
Safety Mechanism
To ensure a controlled replication of the engineered phages, the major capsid protein of the phages was cloned into a separate plasmid to be transformed into the production strain. We added all the necessary parts to create this plasmid into the iGEM registry and combined them to a composite part with existing parts from the registry.
Disclaimer: The promoter we used to create the finished safety plasmid in the registry is not the one we used for our experiments. The promoter we used is also a variant of the T7 Lac promoter that is very well characterized in our facilities. However, it contains sequences that are not compatible with the iGEM registry. As we did not want to mutate this promoter without being able to characterize its mutations, we decided to use the promoter BBa_K2406020 for our contribution to the iGEM registry. In its function and sequence it is very similar to the promoter we used, it is already characterized in the registry and compatible so other teams can use our plasmid.
Next Steps
Even though we were not able to produce our recombinant bacteriophage in time, we still planned the next steps in our experiments with the phage:
- Engineering of the production strain
- Recombination of Phage Genome
- His-Tag purification, Western Blots and ELISAs as Proof of Concept
- Immunoassays with Macrophages and/or Neutrophils
These experiments need to be conducted and evaluated following the design cycle of Research → Imagine → Design → Build → Test → Learn → Improve → Research… before further steps can be predicted.
- Engineering of the production strain
- Recombination of Phage Genome
- His-Tag purification, Western Blots and ELISAs as Proof of Concept
- Immunoassays with Macrophages and/or Neutrophils
These experiments need to be conducted and evaluated following the design cycle of Research → Imagine → Design → Build → Test → Learn → Improve → Research… before further steps can be predicted.
Results
Assembly of the Lambda RED Recombination System
The major part of our work in the wet lab was the assembly of a plasmid containing the Lambda RED system. The left picture shows the result of the PstI digested recombination plasmid (lane number 5). The bands can be seen at the expected sizes (5596 bp and 4143 bp). Furthermore, in lane 3 and 4 the BsaI digested plasmid for our safety mechanism can be seen at the expected positions (2928 bp and 2175 bp).
The picture on the right shows the systematic overview of the Lambda Red System in the Recombination Plasmid.
Production Strain
The designed phage lacks its Major Capsid Protein 10. Therefore, the phage can be cultivated on a strain which supplements the Major Capsid Protein 10. Hence, an open reading frame including a lac-promoter was designed, assembled via Golden Gate cloning and transformed in E.coli BL21(DE3) which produces the Major Capsid Protein 10.
Production of recombineering substrates
To use the Lambda RED system for homologous recombination, a substrate is needed. Therefore, the Protein Human Plasma Gelsolin, a synthetic antimicrobial peptide and the binding site of Human Plasma Gelsolin to LPS (AA 160-169) were amplified via PCR. The picture below shows the result of the amplification. In lane 2 human plasma gelsolin can be seen, in lane 4 the synthetic peptide and in lane 5 the human plasma gelsolin derived peptide.
After that, the products were desalted for electroporation with “Monarch® PCR & DNA Clean-up Kit“. The other substrate for engineering the T7 Phage is of course its own DNA. Therefore, the Phage was cultivated, purified via a PEG-precipitation and the DNA extracted via a Phenol-Chloroform extraction.16 To prove the functionality of the phage DNA, it was electroporated in E. coli BL21(DE3). Unfortunately, no consistent plaque growth was monitored.
Modeling
To conduct the characterization experiments and to produce phage DNA for recombination, phages are needed. These are produced in bacteria batch cultures. To follow the question of how to produce as many phages as possible, a simple modeling experiment was performed. The model concluded that the highest phage concentration can be achieved when introducing the phages to the bacterial culture 40 – 50 minutes after inoculation (see figure below).
16Sayers, J. R., and F. Eckstein. ‘Properties of Overexpressed Phage T5 D15 Exonuclease. Similarities with Escherichia Coli DNA Polymerase I 5’-3’ Exonuclease’. The Journal of Biological Chemistry 265, no. 30 (25 October 1990): 18311–17.
The major part of our work in the wet lab was the assembly of a plasmid containing the Lambda RED system. The left picture shows the result of the PstI digested recombination plasmid (lane number 5). The bands can be seen at the expected sizes (5596 bp and 4143 bp). Furthermore, in lane 3 and 4 the BsaI digested plasmid for our safety mechanism can be seen at the expected positions (2928 bp and 2175 bp).
The picture on the right shows the systematic overview of the Lambda Red System in the Recombination Plasmid.
Production Strain
The designed phage lacks its Major Capsid Protein 10. Therefore, the phage can be cultivated on a strain which supplements the Major Capsid Protein 10. Hence, an open reading frame including a lac-promoter was designed, assembled via Golden Gate cloning and transformed in E.coli BL21(DE3) which produces the Major Capsid Protein 10.
Production of recombineering substrates
To use the Lambda RED system for homologous recombination, a substrate is needed. Therefore, the Protein Human Plasma Gelsolin, a synthetic antimicrobial peptide and the binding site of Human Plasma Gelsolin to LPS (AA 160-169) were amplified via PCR. The picture below shows the result of the amplification. In lane 2 human plasma gelsolin can be seen, in lane 4 the synthetic peptide and in lane 5 the human plasma gelsolin derived peptide.
After that, the products were desalted for electroporation with “Monarch® PCR & DNA Clean-up Kit“. The other substrate for engineering the T7 Phage is of course its own DNA. Therefore, the Phage was cultivated, purified via a PEG-precipitation and the DNA extracted via a Phenol-Chloroform extraction.16 To prove the functionality of the phage DNA, it was electroporated in E. coli BL21(DE3). Unfortunately, no consistent plaque growth was monitored.
Modeling
To conduct the characterization experiments and to produce phage DNA for recombination, phages are needed. These are produced in bacteria batch cultures. To follow the question of how to produce as many phages as possible, a simple modeling experiment was performed. The model concluded that the highest phage concentration can be achieved when introducing the phages to the bacterial culture 40 – 50 minutes after inoculation (see figure below).
16Sayers, J. R., and F. Eckstein. ‘Properties of Overexpressed Phage T5 D15 Exonuclease. Similarities with Escherichia Coli DNA Polymerase I 5’-3’ Exonuclease’. The Journal of Biological Chemistry 265, no. 30 (25 October 1990): 18311–17.
References and Acknowledgements
Our project would not have been possible without the contributions of many people both inside as well as outside of the iGEM support frame. We therefore would like to thank the following people and honor them for their support:
-Michael Sauer, Ass.Prof. Dipl.Natw. ETH FH-Prof. Dr.
-Brigitte Gasser, Assoc. Prof. Dipl.-Ing. Dr.
-Alexandra Graf, Dr.
-Jürgen Zanghellini, Dipl.-Ing. Dr.
-Hans Marx, Dipl.-Ing. Dr.
-Theodor Simak
-Birgit Marckhgott, Mag.rer.nat
-Franziska Doleschal
-Ursula Kiesswetter
-Christine Prenner, Dipl.-Ing Dr.
-Karin Frühwirth
-Christiane Veit, Dipl.-Ing.
-Gabriele Wilt
-MD. Dr. Gattringer
-Dr. Alexander Belcredi from Phagomed
-Dr. med. vet. Sandra Wienhold
-Markus Aleschko, PhD and the staff of Biomin
-BOKU kindergarten
-elementary school Brünnerstraße 139, 1210 Vienna
-Sophia Horak
-Rafael Starnberger
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