iGEM 2024 — Team okstate

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#### Molecular Cloning Notebook

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##### June:

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- Initial experiments are to isolate our target DNA fragments so that we can combine them all in a golden gate assembly. All the combined parts will make up the helper phage that can bind to *S. aureus.*

* Extraction of the M13 bacteriophage plasmid
* Making 5X TBE Buffer stock
* PCR three of the helper phage genes of interest

+ Ytk CamR
+ M13 g2
+ M13 g6

![](https://static.igem.wiki/teams/5444/notebook/1.png)

* Purify the gel extractions of the DNA fragments

+ Check the DNA concentrations:

- Ytk CamR- 46.12 ng/ul
- M13 g2- 36.9 ng/ul
- M13 g6- 12.8 ng/ul

* Test compentency of chemically competent cells using a Ytk plasmid

+ Plating on LB produced a lawn of cells
+ Cells are competent

* Golden gate reactions

+ Ytk + gblocks
+ Ytk CamR + M13 g2 + M13 g6

* Chemical transformations into NEB5a

+ Ytk plasmid + gblocks
+ Ytk plasmid + Ytk CamR + M13 g2 + M13 g6

* Quadrant streaked transformed colonies onto carbenicillin

+ Ytk plasmid + Ytk CamR + M13 g2 + M13 g6

##### July:

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Some genomic regions still need to be isolated and assembled into the final helper phage. The experiments used are to get those parts and preserve the golden gated parts transformed into *E. coli.*

* Make new liquid LB stock
* Freezer stock NEB5a Ytk plasmid + Ytk CamR + M13 g2 + M13 g6
* PCR the remaining genomic parts

+ PA15

* Redo the first three PCR products to get more genomic DNA

+ Running into PCR failures

![](https://static.igem.wiki/teams/5444/notebook/2.png)

![](https://static.igem.wiki/teams/5444/notebook/4.png)

* Found out the thermocycler was not working properly
* Extract the plasmids for the twist parts

+ Plasmid ori
+ M13 ori

##### August:

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* Freezer stock of transformed parts got destroyed

##### September:

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Try and redo all the parts we lost to get the gp3 and gp8 genes of the helper phage.

* Amplified the gp3 and gp8 genomic regions of the main phage

#### Phage Testing Notebook

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##### May

M13 Overlay Assay F+ host: May 28

* Did a standard agar overlay to plate phages and understand M13 plaque morphology

+ Top Images are the view from the bottom of the plate
+ Bottom Images are the top of the plates
+ This procedure was repeated for a while and lysate was harvested from each (this accounts for the time gaps between pictures)

![](https://static.igem.wiki/teams/5444/notebook/bacteria1.png)

##### June

Spot Assay Using Phage Lysate Harvested June 18 To Estimate Phage Titer: June 21

* Did a standard agar overlay to plate phages and understand M13 plaque morphology

+ Top Images are the view from the bottom of the plate
+ Bottom Images are the top of the plates
+ This procedure was repeated for a while and lysate was harvested from each (this accounts for the time gaps between pictures)

![](https://static.igem.wiki/teams/5444/notebook/bacteria2.png)

##### July

M13/K-12 Attachment Assay with Diluted Phage Lysate: July 16/17

* Standard agar overlay but the phage lysate harvested from the plates above was diluted to 10^-6 pfu using TMG as diluent

+ The black dots mark individual plaques and the total counts (approximate) are written on the plates
+ I am not entirely sure why the phage counts lessened, this may be due to different attachment times, or the phage lysate may have gotten too hot and killed some of the phages.
+ 10 minute attachment time before plating

- The overlays below the top three use K-12 as the replacement for the M13 is harvested from using F+ as the host and the diluted phage lysate.
- Attachment time may vary between M13 and K-12, both variations of *E.coli*.

This was done to monitor the phage activity using a bacteria that is different from the bacteria used that produces the M13 phage to try and predict the phage activity if S. aureus was used instead of *E. coli*.

![](https://static.igem.wiki/teams/5444/notebook/bacteria3.png)

##### August

M13/K-12 Attachment Assay with Diluted Phage Lysate: August 8

* Phage count is still inconsistent, but it seems as though the phages prefer to use F+ as the pelleted bacteria compared to K-12, F+ is still used as the host for both, instead of M13 being pelleted and resuspended and chloroformed, K-12 is used.

+ These findings may suggest a differing attachment time that is longer than 10 minutes between *E. coli* strains.

![](https://static.igem.wiki/teams/5444/notebook/bacteria4.png)

Spot Assay with Undiluted Phage Lysate: August 8

* negative control where F+ is added to the top agar and plated, allowed to cool, and then is spotted with 2 uL of undiluted phage lysate, this is done to test that the lysate has not lost pfu’s, we did this to see if that was why we had inconsistent plaque counts.
* A large plaque is seen in the middle of the plate, this affirms that the lysate is highly concentrated and that if pfu’s were lost, it was a minimal amount.

![](https://static.igem.wiki/teams/5444/notebook/bacteria5.png)

M13 Attachment Assay with Diluted Phage Lysate: August 14

* The attachment time was manipulated to see if there were more plaques with a longer attachment time or if the number remained, in this trial we found the highest attachment pfu count to be around 50 minutes
* Attachments longer than 5 minutes become very high. As written above, this heightens the phage’s virulence and range of hosts.

+ We decided to look at attachment times under 5 minutes to prevent this, see information below.

![](https://static.igem.wiki/teams/5444/notebook/bacteria7.png)

Attachment Assay (1- 5 minute attachment): August 19

* An attachment assay with M13 using F+ as a host where the attachment times are in one-minute intervals starting at one minute and ending at 5 minutes.
* After the first minute, there were no plaque increases. The 1-minute attachment had relatively the same amount of plaques as the 5-minute attachment so the increasing attachment intervals appeared to have the same amount of pfus as the initial 1-minute attachment.

+ This suggests that M13 binds to the phage lysate within the span of 1 minute when plated after 5 minutes. We found that anything higher than 5 minutes had too high of a phage titer to be entirely safe due to the virulence risk of the bacteriophage.
+ This is important for understanding how long the phage/dye mixture would have to be exposed to infected skin in order for the phages to be utilized efficiently to target and kill only specific bacteria.

Attachment Assay with *S. aureus*: August 29

* A standard attachment assay where, instead of M13 or K-12 used, *S. aureus* is used and F+ remains used as the host.
* Faint, not completely clear plaques are observed showing that there is phage attachment with *S. aureus* within 5 minutes

+ Not certain that these results provide an accurate portrayal of *S. aureus* phage attachment as there was remaining bacteria in the supernantant that caused the plates to have a very foul odor similar to spoiled milk

- This may account for the turbid plaques

![](https://static.igem.wiki/teams/5444/notebook/bacteria8.png)

##### September

Spot Assay Dilution using F+ as host and Phage diluted with TMG: September 3

* This was done to monitor phage activity, we found that anything lower than 1.2 x 10^6 did not have any plaques
* 2 uL of phage lysate was used and diluted, this was done to count the pfu for another phage harvest, we found that our concentration was relatively the same as the first harvest.

+ This may be because of the controlled OD 600 dilutions of the bacteria before being pelleted for supernatant

![](https://static.igem.wiki/teams/5444/notebook/bacteria9.png)

Attachment Assay with *S. aureus*: September 17

* A standard attachment assay where, instead of M13 or K-12 used, *S. aureus* is used and F+ remains used as the host.
* The attachment, once again, was done allowing 1-5 minutes for attachment.
* No plaques were observed on these plates, some areas where the top agar formed a bubble and then popped the bubble may be confused as plaques.

+ The reason for this is unknown and this procedure was repeated three separate times the first one having residual bacteria in the supernatant causing the plates to spoil.
+ The second two times there were no/faint observable plaques, this may be due to excess chloroform being mixed with the supernatant, killing host bacteria though a lawn appears to have formed.
+ The 5 minute attachment

* Results obtained using *S. aureus* proved inconclusive though we have evidence that M13 bacteriophages have the ability to bind to other bacteria within the *E. coli* variants.

![](https://static.igem.wiki/teams/5444/notebook/bacteria10.png)

## Collective Procedures

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#### 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](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27parts%27%29%20%7D%7D)). 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.

![](https://static.igem.wiki/teams/5444/descriptions-page/pic1.png)

**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.

## References

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1. Kourtis, A. P. (2019). Vital Signs: Epidemiology and Recent Trends in Methicillin-Resistant and in Methicillin-Susceptible Staphylococcus aureus Bloodstream Infections — United States. MMWR. *Morbidity and Mortality Weekly Report*, 68. https://doi.org/10.15585/mmwr.mm6809e1
2. David, M. Z., & Daum, R. S. (2017). Treatment of Staphylococcus aureus Infections. In F. Bagnoli, R. Rappuoli, & G. Grandi (Eds.), *Staphylococcus aureus: Microbiology, Pathology, Immunology, Therapy and Prophylaxis* (pp. 325–383). Springer International Publishing. https://doi.org/10.1007/82\_2017\_42
3. Petrosino, A., Saporetti, R., Starinieri, F., Sarti, E., Ulfo, L., Boselli, L., Cantelli, A., Morini, A., Zadran, S. K., Zuccheri, G., Pasquini, Z., Di Giosia, M., Prodi, L., Pompa, P. P., Costantini, P. E., Calvaresi, M., & Danielli, A. (2023). A modular phage vector platform for targeted photodynamic therapy of Gram-negative bacterial pathogens. *iScience*, 26(10), 108032. https://doi.org/10.1016/j.isci.2023.108032
4. Dubois, C., Atkinson, B., Furie, B., & Furie, B. (2007). Real-Time In Vivo Imaging of Platelets During Thrombus Formation. In A. D. Michelson (Ed.), *Platelets (Second Edition)* (pp. 611–626). Academic Press. https://doi.org/10.1016/B978-012369367-9/50796-5
5. De Plano, L. M., Carnazza, S., Messina, G. M. L., Rizzo, M. G., Marletta, G., & Guglielmino, S. P. P. (2017). Specific and selective probes for *Staphylococcus aureus* from phage-displayed random peptide libraries. *Colloids and Surfaces B: Biointerfaces*, 157, 473–480. https://doi.org/10.1016/j.colsurfb.2017.05.081
6. Tian, L., Jackson, K., Chan, M., Saif, A., He, L., Didar, T. F., & Hosseinidoust, Z. (2022). Phage display for the detection, analysis, disinfection, and prevention of Staphylococcus aureus. *Smart Medicine*, 1(1), e20220015. https://doi.org/10.1002/SMMD.20220015

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## Table of Contents

* [Section 1](#section1)
* [Section 2](#section2)
* [Section 3](#section3)
* [Section 4](#section4)

#### Section 1

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#### Section 2

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#### Section 3

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#### Section 4

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## What should this page contain?

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* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

## Describe what your results mean

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* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](https://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

## Project Achievements

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You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project and what you learned from them. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort. Furthermore, you can still impress the judges by thoughtfully discussing what went wrong and how you might change things.

## Inspirations

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* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)

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