iGEM 2023 — Team usc

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#### November-May

## Overview

Scouring the scientific world to see what issues needed to be solved, we came across an issue right on our doorstep: plant diseases and their impact on Californian orchards.

During our brainstorming process, we hosted weekly general meetings to discuss any potential topics we had discovered and to give overviews of important synthetic biology techniques to get new members acclimated.

#### June

## June 14,2023

After developing our brainstorm into a plausible project, we met with our PhD mentors to see how we could best implement our ideas into action with synthetic biology.

Our potential plan is to create a gene cassette that will incorporate our designed hooks based on the phage genome. Using TAR cloning, we will be able to recombine a BAC/YAC vector with the hooks attached to the cut phage genome. This will allow us to clone our cassette into yeast.

A BAC/YAC vector contains bacterial and yeast DNA and origins of replication. It also contains a yeast centromere and selectable markers for yeast. Yeast has high levels of homologous recombination, making it the best host organism to house our plasmid with the vector and gene cassette.

## June 16-25,2023

After searching through many articles, we found the annotated genome for our bacteriophage of interest, phage phi EA104 on NCBI's database. Nikky uploaded this onto Benchling and we got started on designing guide RNAs and hooks based on Benchling's suggestion and our hopes for genetic engineering.

Using Benchling's software and our PhD mentors' guidance, we began looking for an essential phage gene to delete!

#### July

## July 6, 2023

Kelsey, Dani, and Nikky met with Carol Bornstein, a Horticulturist and Botanist that has been working with California's native flora and fauna for decades.

This meeting gave us important insight into how experts tackle plant diseases when they show up using the Integrate Pest Management protocol. Learn more about what we learned from Carol Bornstein in our Human Practices section!

## July 19& 20, 2023

Starting wetlab experimentation, we did PCRs of both our sgRNA an Hooks that were designed and ordered specfically from Twist Biosciences and IDT.

These PCRs followed the standard workflow that our host lab, the Ehrenreich lab at USC, performs. After running gel electrophoresis to verify that the PCRs were successful, we ran into an issue. The gel did not meet our expectations, with the undiluted hooks being smeared and the diluted hooks absent.

## July 24, 2023

Using a Zymo Kit, Nikky, Dani, and Kelsey completed DNA purification on the sgRNA and hooks that underwent PCR. After using the kit, the DNA was quantified with a Qubit machine, which verified that we had sufficient DNA concentrations to move forward.

DNA purification was followed by a T7 Expression to make our hooks into useable RNA. After following standard T7 expression protocol, we incubated overnight at 37°C.

## July 31, 2023

When considering next steps in our experimental procedure, we discovered that we would need to use Beef Extract Nutrient Broth in order to work with the phage DNA ordered from ATCC. In the mean time, our goals are to:

1. Identify a gene required for replication of the virus.
2. Identify the sequence of said gene and primers to amplify it.
3. Find a location in the bacterial genome to which we can clone it.
4. Identify Cas9 sgRNA sites which can target the gene in the viral genome.

#### August

## August 15-17,2023

Using the NEB Phage Prep and Plating protocol, we made 8 soft agar plates of varying dilutions [1x-1000x] of the propagated bacteriophage to test the phage's efficiency.

After allowing the plates to grow overnight, we believed we observed the formation of plaques---these show where the phage successfully killed the E. amylovora bacteria on the plates. We selected the best four plates that showed cell death visible to the naked eye and combined them to create one lysate.

![](https://static.igem.wiki/teams/4857/wiki/2.jpg)

## August 18,2023

Using the NEB HMW Monarch DNA Extraction kit, we began with 300µL of lysate and followed the protocol omitting the step to resuspend in 5mM MgSO4. At the end, we were left with low DNA concentration as read by both the Qubit and NanoDrop.

We will attempt to use this kit again following the protocol exactly to see if we can obtain higher DNA concentrations.

After quantifying with a Qubit, there was no DNA concentration. Thus, we will proceed with different steps.

## August 22,2023

Because there was an issue with the DNA concentration, we decided to attempt to replate the virus and bacteria to form plaques. However, this time, we waited too long before pouring the soft agar and it solidified in the 15mL tube.

Thus, there was only enough pre-made soft agar to make 3 plates, to which these dilutions of the phage were added:

1. New 1x
2. From previous lysate 1x
3. From previous lysate 1:10

When replicating, don't forget to pour soft agar right away after adding bacteria!!!

## August 28,2023

After plating the previous week, we decieded to try a liquid outgrowth from the glycerol stock of the bacteria. It was then given 50µL of the phage to test OD and see if the phage and bacteria interact.

After 1 hour of incubation, there was an absorbance of 0 from the spectrophotometer. This means that there is no initial volume at innoculation. After checking with a NanoDrop, we decided there was no DNA concentration and the outgrowth did not work.

Our next plan of action is to incubate overnight to have an overnight culture and to do the New England Biolabs extraction protocol using MgSO4

#### September

## September 11,2023

Todays goals were to remake the lysates and plate them.

Next steps include:

1. Quantify/measure collected lysate with Qubit
2. Create PEG + NaCl
3. Incubate overnight
4. Centrifuge and Resuspend in SM Buffer

## September 12,2023

We had successfully lacey plates at the 1:1,000 and 1:10,000 dilutions, thus we will make 4 plates of the 1:1,000 and 2 of the 1:10,000 dilution to make new lysate for increased phage in supply.

Next steps include making a new lysate off new plates and performing phage precipitation to see side by side which works best.

## September 14 &15,2023

Today we made new lysate from the plates.

After performing the High Molecular Weight Extraction on the lysate and precipitate, we were able to get no DNA from either protocol. We quantified with a Qubit.

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## Process Overview

Despite our results attempting to propagate the phage, the experimental process below outlines our projected methods. After propagating the phi EA104 bacteriophage, we would have used the following methods:

## Steps

1. - ### In Silica Work

   * We used the annotated phi EA 104 phage under the Kolesnikvirus taxonomic family and place it into Benchling after downloading from NCBI.

   * Next, we deisgned a gene cassette focused on cloning the phage as an insert into a BAC/YAC capture vector in order to grow the plasmid in E.coli. Our end goal is to cut out a gene essential for the maturation of the phage genome, yet will still allow already developed phages to infect our target bacteria. This aims to make it so that only phages propagated in a lab setting can fully develop into a mature form. This biocontains the phages that have been released into the environment to ensure that they do not replicate and overtake other microbes once the target bacteria (E.amylovora) has been eradicated.

   * Our gene cassette used guide RNAs and arms of homology (hooks) to use the power of homologous recombination with the vector in yeast.
   * The BAC/YAC vector works when the insert [in our case the phage genome] and reagents needed for cloning are transformed into yeast. The pcc1BAC vector is designed with a **YAC** that has a yeast centromere, selectable marker for yeast, and an origin of replicaiton. It also contains a **BAC** portion with a multicopy and single copy replication origin, a marker, and a control segment.
   * Yeast have high levels of homologous recombination. Thus, if the BAC/YAC vector and the desired insert are in proximity within the yeast cell with arms of homology, the insert will be able to recomine.

   Fluid Image Example

   ![Fig 1 sept7 slides](https://static.igem.wiki/teams/4857/wiki/2.jpg)
   ![Fig1.2 pcc1bac diagram](https://static.igem.wiki/teams/4857/wiki/a.jpg)
   ![Fig 1.3 hooks and guides](https://static.igem.wiki/teams/4857/wiki/screen-shot-2023-10-11-at-10-47-53-pm.png)

   * In the yeast nucleus, the target phage portal protein can be deleted by co-transforming in the guide RNAS designed. The guide RNAs are designed specifically with CRISPR in mind. A repair template with a selectable marker to 60bp single strand of DNA will be used to replace the deleted.

## Conclusion

As we focused more on the phage propagation aspect,our engineering aspects were not as developed as we would have liked.

## Phage Propagation

1. The phi EA 104 phage was ordered from ATCC. The protocol for propagation was followed as directed by the website.

   * Prepare actively growth culture of host strain in the recommended media. Allow to grow, shaking for 18-24hrs (overnight) at 26Cº.
   * Add 1mL of the recommended broth to the freeze-dried phage, and .5mL to a cry-vial.
   * Prewarm plates in incubator, overlay the solid plates with soft agar media (.5% agar) which has been inoculated with 1-2drops of the host culture.
   * Prepare dilutions of the resuspended phage by moving 100uL of the suspension into 900uL of media (1:10) and serial passage as needed to prepare additional dilutions.
   * Once the soft agar overlay has hardened, add dilutions of the page, dropwise, to the plates. A new plate should be prepared for every dilution of phage desired. Add the phage to the soft agar prior to pouring and hardening.
   * After 24hrs of growth, scrape off the soft agar and centrifuge at 1000rpm for 25min to pellet the cellular debris and the soft agar.
   * Save the supernatant and pass through a .22 filter and save the lysate at -20C.

   When plaques are formed and identified, 5ml of nutrient broth is added to each plate to do overnight growth once again. The next day, the soft agar and liquid is scraped into the 15ml tube, centrifuged at the conditions specified above in the ATCC protocol, and filtered to make the lysate.

   ![Fig 2](https://static.igem.wiki/teams/4857/wiki/fig-2.jpg)
2. ### Genomic Extraction

   Using the New England Biolabs High Molecular Weight DNA Extraction Kit, the phage DNA was attempted to undergo isolation.

   - - Centrifuge the culture at 12000g for 25min to pellet the cellular debris.
     - Next, precipitate phages by incubating the filtered supernatant with 10% PEG and 1M NaCl at 4Cº to overnight.
     - Precipitated phages were collected by centrifugation at 12000g for 10min. Remove the supernatant.
     - Resuspend the phage in 50 mM Tris-HCl pH 7.5 buffer, 10 mM MgCl , and 100mM NaCl.
     - The phage was lysed using 300uL of the HWM gDNA Tissue Lysis Buffer and 20uL of proteinase K for 45 minutes at 56ºC shaking at 300rpm
     - 300uL protein separation solution was added and gently mixed. Centrifuge at 32000g for 10min.
     - Transfer the resulting (top) aqueous layer to a new tube. Add two DNA capture beads.
     - DNA precipitation was initiated by adding 550uL isopropanol. Slowly, gently, manually invert 25 times.
     - Remove the supernatant by pipetting carefully to not disturb the DNA bound to beads.Wash 2x with 500uL gDNA wash buffer, removing the wash buffer in the same way as above.
     - Pour beads into the provided container and pulse in a microcentrifuge to remove residual wash buffer.
     - Add the DNA beads to 100uL of gDNA elution buffer II and incubate at 56ºC for 5minutes to allow the DNA to be released from the beads
     - Transfer to a bead retainer to separate the beads from DNA by centrifugation at 12000g for 1min.

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   After various iterations of the genomic extraction protocol ranging from trying to omit steps from the NEB protocol to fulfilling every step as listed, there was not sufficient DNA concentration to work with in planned experimental methods. Thus, we were not able to move forward with our cloning project and determined that the phage selected is not robust enough in a lab setting to be manipulated for a product that would be able to be used in orchards

## Conclusion

Bacteria and phage were not compatible by way of current experimental design methods in terms of safety protocols. Next steps would be to use phenylchloroform, which is a highly dangerous carcinogen.Projected targets for genomic extraction from DNA not reached due to time constraints after trying various methods to get reasonable DNA concentrations to continue experimentation.

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## Overview

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Portal proteins exist within the Erwinia phage genome with portal complexes that have different contortions per each stage of phage assembly (Prevelige, 1993). This potential target protein is key in capsid assembly and the DNA packing process (Dedeo, 2019). Potential infection and bacteriophage development catalyzation are directly corresponded to these portal proteins. Further experimental research and redesign would allow for the exploration of deleting this gene from the bacteriophage genome to regulate the progression of infection. Another potential direction that could be further explored includes induction of anaerobic respiration to potentially reduce the ability of E. amylovora to survive. E. amylovora have genes involved in anaerobic respiration, and during these metabolic processes, the genome size can become reduced or experience loss and decomposition of genes responsible for growth cycles (Sebaihia, 2010). To further test and examine these phenotypic-genotypic relationships, future directions include using conditions of limited oxygen supply cultured in a hypersaline environment to describe bacteriophage progression when in anaerobic conditions (Muller, 2005).

Although this project was unable to successfully eliminate the phiEa104 bacteriophage by way of Erwinia amylovora bacteriophage genomic extraction, this investigation elucidated potential new target sites, replacements for pesticides, and ideas for biocontainment tactics against microbial attacks. As mentioned above, our team has explored other potential avenues for targeting genomic deletion to mitigate bacteriophage assembly. Hypothetical implications corroborate the necessity of development within fields of containing bacterial infection among crops. Furthermore, based on our formulative research, targeting proteins that control the morphology of the virus could mediate growth phases and assembly of viral infection. Ultimately, cloning techniques, genomic extraction, and the power of homologous recombination have the potential to eliminate Erwinia amylovora as a threat entirely.

## Project Achievements

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* Inoculated bacterial liquid culture with virus for genomic preparation.
* Effectively carried out viral infection of liquid cultures to generate viral lysate for genomic extraction.
* Successfully produced viral plates containing bacterial cultures that had phage plaques.
* Identified viral plaque formation on plates.
* Able to obtain DNA via genomic extraction of phage after outgrowth.

## Unsuccessful Segments

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Ultimately, the Erwinia amylovora bacteria and specified phage do not interact to a desirable level for utilization as hypothesized.

* Bacteria and phage not compatible by way of current experimental design methods in terms of safety protocols. Next steps would be to use phenylchloroform, which is a highly dangerous carcinogen.
* Projected targets for genomic extraction from DNA not reached due to time constraints after trying various methods to get reasonable DNA concentrations to continue experimentation.

## Sources

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* Dedeo, C. (2019, September 29). Portal protein: The orchestrator of capsid assembly for the dsdna tailed bacteriophages and herpesviruses. National Library of Medicine - NCBI. <//www.ncbi.nlm.nih.gov/pmc/articles/PMC6947915/>
* Muller, J. (2005, March). Genomic Analysis of Anaerobic Respiration in the Archaeon Halobacterium sp. Strain NRC-1: Dimethyl Sulfoxide and Trimethylamine N-Oxide as Terminal Electron Acceptors. National Library of Medicine.  [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1064022/](//www.ncbi.nlm.nih.gov/pmc/articles/PMC1064022/)
* Prevelige, P., & Thomas, D. (1993, March.). Nucleation and growth phases in the polymerization of coat and scaffolding subunits into icosahedral procapsid shells. National Library of Medicine - NCBI. <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1262396/>
* Sebaihia, M. (2010, April 1). Complete Genome Sequence of the Plant Pathogen Erwinia amylovora Strain ATCC 49946. Journal of Bacteriology. <https://journals.asm.org/doi/10.1128/jb.00022-10>

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