Wet Lab Engineering Success

Wet lab Design and Engineering Success

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Throughout our work on Healios we encountered several setbacks, forcing us to step back and reassess our approach to developing the current iteration of Healios. Through several rounds of trial and error, we have arrived at a process that works effectively and suits our purposes.

Project Motivation

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There is currently a large medical issue regarding Staphylococcus aureus, a bacteria capable of causing invasive infections, sepsis, and death. It is one of the most common pathogens in healthcare facilities and in the community. ^[1]

Why is this a problem?

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In 2017 there were nearly 120,000 cases of S. aureus bloodstream infections, and nearly 20,000 deaths, comprising both Methicillin-resistant S. aureus (MRSA) and Methicillin Susceptible S. aureus (MSSA). ^[1] Currently there are few effective treatments that can target these S. aureus isolates without the use of multiple antibiotics^[2].

Where do we start?

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We first had to come up with something that could attack these S. aureus isolates without further harming the patient. This is when one of our advising professors, Dr. George Huang, informed our team about the possible use of photodynamic therapy and bacteriophages chemically conjugated with a photoreactive dye.^[3] We decided to make the creation of these chemically conjugated phages the focus of Healios. If we could engineer a phage capable of binding to S. aureus and chemically conjugate this phage with a photosensitizer such as Rose Bengal (RB), which generates reactive oxygen species when exposed to green or blue light (540 nm)^[4], then we would have a targeted treatment capable of killing S. aureus without harming the host tissues in the process. ^[3]

Making a plan:

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The first step in creating this form of targeted S. aureus treatment is selecting an appropriate bacteriophage, one capable of both conjugating with Rose Bengal and effectively binding to S. aureus, either naturally or through modification. Our team decided on using M13, a bacteriophage with a capsid capable of conjugating with hundreds of Rose Bengal photosensitizers.^[3] The M13 bacteriophage can also be modified to bind to S. aureus,^[3] making it the ideal bacteriophage chassis for our project.

Cycle 1

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After deciding upon using M13, we needed to find a way to produce the modified M13 necessary for S. aureus treatment. We set our sights on the first requirement of the modified M13, S. aureus binding. To allow M13 to bind to S. aureus we would need to modify the phage’s capsid with a binding structure specifically for S. aureus. Gp3 and Gp8 are the two capsid structures that we selected for modification. As the natural Gp3 structure of M13 targets E. coli, we will need to modify it to target S. aureus. E. coli was, naturally, the best choice for synthesizing our modified M13 phages as E. coli is the natural host of M13. By searching through the literature, we identified multiple studies where the researchers used a technology called phage display to identify peptide sequences capable of binding specifically to S. aureus.^[5–7] We decided on three peptides (See Parts for more details). As a production backbone, we constructed a plasmid containing the low copy PA15 origin of replication, carbenicillin resistance gene, an f1 origin of replication, and a BsaI cloning site upstream of either gp3 or gp8. This would be plasmid 1. Another design idea we utilized was that if we only used a one plasmid system to express all the phage proteins, we would get very few phage particles made. Therefore, plasmid 1 would only be making Gp3 or Gp8 and the plasmid would encode for a phage packaging signal. Plasmid 2 will be the helper phage, which will encode for all other phage proteins but lacks a phage origin of replication. Thus plasmid 2 would be responsible for making all the other structural phage proteins without the genome being packaged.

Figure 1. Schematic detailing the two plasmid systems for S. aureus targeting M13 phage.

Our cloning strategy then involves assembling a constitutive promoter followed by a ribosomal binding site, a DsbA signaling peptide, followed by one of the three S. aureus targeting peptides, and lastly the in-frame chimeric fusion of the peptide with gp3/8. The secretion peptide is necessary to ensure the translated chimeric protein can translocate across the E. coli cell membrane via the Sec secretion system. The entire construct can be assembled with Golden Gate cloning with BsaI sites. Our system also allows for the swapping of any other bacterial-specific targeting peptide to Gp3/Gp8 easily.^[3] The end results would be a product of non-infective phage particles containing S. aureus retargeted peptides that will be capable of binding to the cell wall of S. aureus.

Testing Phase and Problems Encountered

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We ordered the gene blocks from TWIST and IDT. We had two processes that needed to be done. The first was to remove the original Gp3 and Gp8 gene from the helper phage plasmid. The second was to assemble the first plasmid (Gp3/Gp8 production plasmid) using Golden Gate assembly. During the process of performing Golden Gate assembly for the Gp3/Gp8 plasmids, we transformed the plasmids into E. coli but ended up with many colonies and we were not able to identify which one contained our desired plasmid by PCR.

Cycle 2

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We realized that we need to optimize our assembly to allow for an easier screening method for the right donor cassettes. To accomplish this, we changed the parts so that a mRFP dropout cassette would be cloned next to the promoter but with an adjacent BsaI site. During digestion with BsaI, mRFP should fall out allowing for the rest of the parts that would contain the chimeric Gp3/Gp8 biobricks to ligate. Any transformants that turned red would indicate the colonies contained the uncut mRFP, while colonies that were white would indicate the dropout cassette has been removed and the assembly should be correct.

Testing Phase and Problems Encountered

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When we performed our plasmid transformation into E. coli and incubated the cells on an agar plate, we quickly noticed that none of the bacterial colonies were red. This either indicated that the mRFP dropout cassette was 100% successful, or that there was an issue with the gene expression of mRFP. As a 100% successful cassette dropout is extremely unlikely, we looked into the design of our cloning vector to check for any issues. We discovered that the ribosomal binding site was missing. With no RBS, mRFP would never be expressed, meaning that when Golden Gate ligation was periodically unsuccessful the bacteria would not be red, leaving us with no visual indication of whether or not a colony is viable for Healios.

Cycle 3

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The only thing that we could do at this point was reorder the plasmids, this time with the ribosome binding site, and start the whole process from the beginning. This would solve our issue with distinguishing colonies transformed with the proper Golden Gate ligation from colonies who had transformed with unchanged plasmids.

Testing Phase and Problems Encountered

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Having received setbacks in our cloning we eventually received the proper parts with a mRFP dropout cassette and a rbs to drive its translation. We encountered another problem when we were swapping in the phage origin of replication for the production plasmid while removing the wildtype Gp3/Gp8 genes from the helper plasmid. We were not able to get any PCR products even though initial pilot reactions worked. All subsequent PCR reactions did not work although we used the same perimeter. As the deadline for wiki freeze drew near, we eventually switched to a different PCR machine and were finally able to get PCR products. Furthermore, we also ran into the problem of losing our competent cells. The cells were stored in a deep freezer that ended up being decommissioned and we lost all the cells for our transformation reaction.

Possible Future Improvement

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We unfortunately ran out of time for our project. However, we have learned a lot and were able to apply the engineering process in troubleshooting our cloning problems. We are now confident that given more time we will be able to complete the assembly of the two plasmid systems. Importantly, we have demonstrated how predominant the engineering process is in synthetic biology.