Notebook

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.

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!

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!

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.

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.

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.

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.

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.

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!!!

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

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

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.

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.