iGEM 2022 — Team michiganstate

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## Beginning Stages

##### May 31st, 2022: Gathering Our Samples

The team spread out around our beautiful campus and collected different samples in hopes to isolate an environmemntal bacteriophage. We visited the Red Cedar River (1), botanical garden pond (2), Arboretum pond (3) and our on-campus greenhouses (4) to collect water samples from the river/pond and soil samples from DC3000 tomato plants!

![](https://static.igem.wiki/teams/4135/wiki/getimage.jpeg)

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* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

##### June 1st, 2022: Phage Isolation and Amplication

We followed the Phage Isolation Protocol and started the amplification using the second samples from 5/31/22. Amplification was done using normal quantities with an extra ~5mL of KB broth for the Arboretum pond water and Beal Botanical gargen water samples. Each amplification was transferred from the falcon tubes and incubated for 12 hours at 28 degrees Celsius with an rpm of 210.

* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

##### June 2nd, 2022: Phage Environmental Isolation

The Phage Isolation Protocol was followed and each other amplification samples (1-4) was used. The samples were well turbid with growth and we did let them incubate for an extra 2 hours for a total amplification time of 16 hours at 28 degrees Celsius. Filtration was initially attempted using the straight 10mL amplication but we found it extremely difficult especially with the soil samples. To help ease the process, we spun each sample using a centrifuge in 15mL falcon tubes. We spun the samples at 4,000 x RPM for 10 minutes for a nice pellet consisting of the P.Syringae and the soil contaminants. We then used the supernatant for filtration using the 0.22µm millipore filters into 50mL falcon tubes. The new environmental filtrates were then used to make putative plaques. The host amount was changed to be 120µL due to the host being grown for only 6 hours prior that same day. A total of 16 plates were made. The plates were made by mixing the 120

The Phage Isolation Protocol was followed and each other amplification samples (1-4) was used. The samples were well turbid with growth and we did let them incubate for an extra 2 hours for a total amplification time of 16 hours at 28 degrees Celsius. Filtration was initially attempted using the straight 10mL amplication but we found it extremely difficult especially with the soil samples. To help ease the process, we spun each sample using a centrifuge in 15mL falcon tubes. We spun the samples at 4,000 x RPM for 10 minutes for a nice pellet consisting of the P.Syringae and the soil contaminants. We then used the supernatant for filtration using the 0.22µm millipore filters into 50mL falcon tubes. The new environmental filtrates were then used to make putative plaques. The host amount was changed to be 120µL due to the host being grown for only 6 hours prior that same day. A total of 16 plates were made. The plates were made by mixing the 120µL P.Syingae with 100µL CaCl2, 100µL of filtrate all into 1mL of KB broth. The samples were then incubated for 30 minutes at 28 degrees Celsius then plated onto small sterile petri dishes using 7mL of KB agar. 4 plates labeled A-D was done for each sample.

So, the designations on the plate are, for example, 1A-1D for the environmental filtrate plates. Essentially all the plates under each sample filtrate are technical replicate. We did this to ensure we get as many unique plaques as possible and have the opportunity to pick one of each type of plaque morphology. By doing more than one plate we can discard any contaminated or otherwise useless plates. For made new enrichments in the same 10mL KB broth with the same amounts in the Phage Isolation Protocol except with 1mL of the overnight P.Syringae. These samples will be filtered similarly as done today. The rest of the filtrates were capped and stored in 4 degrees Celsius.

* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

##### June 3rd, 2022: Phage Environmental Isolation

The Phage Isolation Protocol was followed and putative plaque screening was done by picking at least one of each unique plaque into an Eppendorf with 1mL KB and 100µL of CaCl2. Plaques picked were labelled using a sample number and letter. Each plaque description was done by Jenna on an Excel sheet.

Two images of the unique plaques we picked are depicted in the following figures

![](https://static.igem.wiki/teams/4135/wiki/igem-notebook/0603pic1.jpg)

Figure 1. Sample 2 (Red Cedar Silt) filtrate plated with P. Syringae. Shown are pin-point sized plaques with fully clear and temperate plaques. Sizes of the plaques also varied but stayed small despite the deviations.

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![](https://static.igem.wiki/teams/4135/wiki/igem-notebook/0603pic2.jpg)

Figure 2. Sample 4 (Tomato plant debris) filtrate plated with P. Syringae. Shown are very large plaques with bullseye type clearing. Each plaque had very clear centers with cloudy hallows. The sizes varied but most of them were consistent with the large type morphology.

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##### June 6th, 2022: Serial Dilution

We used the weekend to incubate the plaque picks and added 25µL of chloroform gently; inverting it to bring any host bacterium to the bottom of the Eppendorfs before making 1:10 serial dilutions ranging from -1 to -7. We did dilutions of 10µL to the plaque pick into 90µL of KB and then did the serial dilutions of 10µL into the 90µL KB. An image is here for easier interpretation.
![](https://static.igem.wiki/teams/4135/wiki/igem-notebook/0606pic1.png)

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Serial dilutions were done in PCR strip tubes. This was difficult to do because of the individual caps. Next time, we will be using proper 96-well plates to do the serial dilutions.

We set aside the serial dilutions and made plates using molten KB agar. We first added 150µL of P. Syringae from a fresh overnight culture into each tube. We then added 8mL of cooled KB agar to each tube and plated them. Following this, we spot plated 5µL of dilutions ranging from -3 to -7 on the plates and labeled which spot dilutions corresponded to the plaque pick. We spotted 3 plaque dilutions per plate with a control that consisted of just host and no spots.

##### June 7th, 2022: Meeting with Mark Forbush

Today, we had a meeting with a representative of Future Farmers of America (FFA), Mark Forbush. The team compiled a list of questions to ask him relating to our iGEM project. The questions we asked him are listed down below:

* What are the current advantages of current commercial pesticides? More specifically the use of metal-based pesticides like copper sprays used?
* What are the disadvantages you see for the use of current commercial pesticides? (This can be economical or crops)? More specifically the use off metal-based pesticides like copper sprays used?
* Have you ever heard about alternative methods of biocontrol and if so, what are they?
* Is there economic or social pressure to use or not to use GMOs in crops?
* Have you ever heard about phage being a type of biocontrol? Do you know what phages are?
* How willing are farmers to cooperate with scientists and industry to create real-time biocontrol solutions?
* How can we increase engagement in the agricultural community?

The responses are documented on a Notion page as well as a recorded Zoom meeting that is saved on James's laptop.

##### June 9th, 2022: Characterization

We were successful in having new plaques on our plates from dilutions -5 to -7. The team started to characterize each plate and were circling the plaques that we wanted to pick off. When we looked at our control plates today, we saw some contamination on both the positive and negative control.

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 ![](https://static.igem.wiki/teams/4135/wiki/igem-notebook/0609pic1.jpeg)

As we were troubleshooting, the other plates with the plaques had no contamination that is seen on the controls. We decided to re-autoclave the agar we had sitting in the water bath and to also make another 1L batch of KB agar.

Today, we are planning to pick plaques and to suspend them in Eppendorfs with 1mL of KB and 200µL of the 500mM CaCl2 and incubate them for 48 hours. The smaller plaques that we have will have 1mL of KB and 100µL of the 500mM CaCl2 and they will also be incubated for 48 hours.

##### June 14th, 2022: Meeting with George Sundin

ask team members to reorganize the notes for this day and add in after

##### June 22nd, 2022: Soft Agar Overlay

Lindsay, a graduate student from George Sundin's lab came in today and taught us the procedure for how to do soft agar overlay. We divided the plates into 6 areas for the different dilutions. This helps us to prevent using so many plates and get us more dilutions on one plate.

These are the steps for the soft agar overlay.

1. Stripette 8mL of KB agar into a 100mm X 15mm plate and let it cool.

2. Heat KB agar and sterile water around half an hour before starting.

3. Spin 1mL of bacteria for 1 minute.

4. Decant the KB broth and resuspend in ~1mL of phage buffer.

5. Add 5mL of sterile water and 5mL of KB agar into a falcon tube.

6. Make sure that before adding in any host that the temperature of the agar and the sterile water is not too hot.

7. When it is lukewarm, pour the contents of the falcon tube onto the KB poured plates.

When doing spot plating on soft agar overlay plates, change the pipette to 25µL and take the different dilutions and spot them on the plates. Let this sit in the fume hood until it has dried.

##### June 23rd - June 27th, 2022: Purification

We repeated the purification steps by doing the soft agar overlay. On June 24th, we were on the last round of purification and we were doing dilutions ranging from 0 to -10 for the big plaques. The smaller plaques were being diluted down to -5. James came up with a labeling system to keep the Eppendorfs in order. We did not use any of the overnight sample because it had not grown enough, but instead we picked directly from one of the plates of P. Syringae and put it into the falcon tube before laying the soft agar overlay on top of the KB agar.

On June 27th, the whole team made dilutions ranging from -3 to -4 for the big plaques. For the small plaques, we diluted down ranging from -2 to -3. There were different techniques that everyone on the team used so that we could see how the different techniques would appear on the plates. For example, Roxy, Anna, and Jenna did soft agar overlay. James and Ram did do soft agar overlay, but instead put the host inside the KB agar directly onto the plate and spot plated 5µL instead of the 25µL that is used on the soft agar overlay.

##### June 28th, 2022: Lysates and SpyCatcher/SpyTag Systems

Today we had a meeting with Lindsay to talk about our future plans. She gave us 50mL of phage buffer to flood our plates. Each of us flooded 2 plates each and there were different protocols we followed to flood them. We are now at the stage where we are making our own lysates.

We had conversations with both Lindsay and Danny Ducat about us modifying our phage to be UV resistant. With Dr. Ducat, we talked about the spycatcher/spytag peptides. We want to use this system so that we can prove we can make modifications on this capsid so that we have proof that we can make modifications before we get to the process of actually putting the spycatcher and tags on. This would help us make models on Chimera with Sundhar using the programs he showed us.

##### June 30th, 2022: Lysates

Today we are spot plating our lysates from dilutions of 0 to -12. Everyone has 2 separate plates each for the different lysate samples that we have.

During our weekly meeting with the mentors, we talked more about outreach/collaboration, social media accounts, and fundraising. We put an emphasis on getting started on those along with our wetlab experiments. We are also having the mentors look over our MID-SURE abstract that is due July 1st. MIDSURE is a mid-Michigan Symposium for undergraduate research experiences. This is a great way for us to practice for the big Jamboree that is upcoming this October.

##### July 5th - July 11th, 2022: Efficiency of Plaquing

Efficiency of Plaquing (EOPs) is used to see the plaque morphology and measures the titer of the phage. We did EOPs and diluted the content ranging from 0 to -12. This was one of our first times doing EOPs so each team member had their own ways of dividing up a plate to spot plate the plaques.

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 ![](https://static.igem.wiki/teams/4135/wiki/igem-notebook/4c74901a-18cd-4d84-a69f-f533cffb57b6.jpeg)

Figure 1. James Suggitt did his EOPs in triplicates replicating all the dilutions in a horizontal manner.

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 ![](https://static.igem.wiki/teams/4135/wiki/igem-notebook/63ce7f83-224f-48ee-97a0-6c4732421a83.jpeg)

Figure 2. Anna Kim did her EOPs in triplicates replicating all the dilutions by dividing up the plate into sections.

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Throughout this week, the team practiced getting familiar with the steps in doing EOPs. Considering that every team member had their own way of dividing up the plates, we decided that we were going to use Figure 1 as our consistent way of measuring the EOPs from here on out.

##### July 12th, 2022: Grants, Growth Curves, & Social Media

Anna has been communicating with the previous iGEM team members for the social media account passwords. She started posting on the iGEM instagram page introducing the team members every day.

As the team members were looking on other iGEM social media accounts, it inspired us to look into ways to crowdfund or use GoFundMe to fundraise for the team. In addition to this, Jenna and Anna are a part of the Lyman Briggs college here within Michigan State University. They both contacted the dean of Lyman Briggs, Kendra Spence Cheruvelil for funding. She responded back and forwarded us to be in contact with Niki Rudolph.

For wetlab events, we are doing a growth curver of the optical density of our overnight culture. We are also using a system called Chimera, Phrye2/Collabfold to look at our models for REC1, SHL2, FRS, and COT4. This system was introduced to us by Sundhar. As a team, we were all figuring out and troubleshooting these systems because we were unfamiliar with how they worked.

##### July 13th, 2022: Grants

The team received great news today! Jenna and Anna got $500 each from the Lyman Briggs College through Michigan State University. This is a great help to our team as we have been searching for ways to be able to fund ourselves for the trip. Thank you Lyman Briggs for supporting iGEM 2022!

##### July 14th, 2022: Chimera and Imaging

The team met with Sundhar and talked about possible placements of where we can put our linker and spytag. Throughout our discussion, we saw on the 3D Chimera model that the N terminus would be difficult to attach because the predictions on the model were not stable. However, the C terminus has consistent modeling throughout all the different chains and the surface was exposed for us to attach the linker and spytag. We are moving forward with trying to model using the C terminus now. We also looked at a codon optimization table to see which codon would be best for us to place it on.

In this meeting, we were also talking about doing some imaging for our phage using our high titer lysates. We planned out a day for some of the team memebers to go to the Cryo-EM facility to do cryo-em imaging of our high titer lysates. Roxy, Anna, and Jenna are going into the lab July 29th with Sundhar to check out the facility and to get images for the team.

##### July 17th: CFU Growth Curve Calculations

Today we counted our CFU plates and did our growth curve calculations in the excel spreadsheet. We took our overnight and made 6 half dilutions and measured the initial OD reading for all the samples. After, we did the standard EOP protocol and diluted the sample down to -12 and plated these on KB agar with host. We grew these overnight and colonies of P. Syringae formed.

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 ![](https://static.igem.wiki/teams/4135/wiki/igem-notebook/8d76be57-1a2f-4912-bd00-2f8d03bd0d8e-1-201-a.jpeg)

Figure 1. CFU plate 1

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 ![](https://static.igem.wiki/teams/4135/wiki/igem-notebook/9c9fc99d-da0b-40c3-bbfc-27334d23a572.jpeg)

Figure 2. CFU plate 2

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 ![](https://static.igem.wiki/teams/4135/wiki/igem-notebook/4ff8e7ed-2641-42bd-90d9-7a4e48db6f92.jpeg)

Figure 3. CFU plate 3

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 ![](https://static.igem.wiki/teams/4135/wiki/igem-notebook/aaa0037f-ca81-4caa-a786-d227b8dbd91c.jpeg)

Figure 4. CFU plate 4

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 ![](https://static.igem.wiki/teams/4135/wiki/igem-notebook/cd245de4-234e-4e7d-97df-330c181f5a53.jpeg)

Figure 5. CFU plate 5

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 ![](https://static.igem.wiki/teams/4135/wiki/igem-notebook/af3da338-d375-454f-98bd-97b521b437ab.jpeg)

Figure 6. CFU plate 6

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We then counted the colonies and we had a preset Excel sheet where we could input our data and get the growth curve calculations. The results are below:

![](https://static.igem.wiki/teams/4135/wiki/igem-notebook/screen-shot-2022-08-17-at-2-50-06-pm.png)

Figure 7. Data from Growth Curve Calculations

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![](https://static.igem.wiki/teams/4135/wiki/igem-notebook/growthcurveset1.png)

Figure 8. Graph from Growth Curve Calculations

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##### July 22nd: MIDSURE Poster

Our team finalized the MIDSURE poster and are going to send it to the PI's to review and give feedback for us. We also found modification targets for the T7 major capsid protein and put them in the DNA targets document on the shared Google Drive. We are also finishing up the pitch that we were writing for when we present. To keep the team more organized, we are also writing a calendar outline for the upcoming weeks and the tasks we need to get done in the wetlab.

##### July 25th: EOP's and Lysate

Today, we did EOP's with the T7 samples on E.coli LB plates. In addition, we confirmed our high titer lysates by spotting 5µL of each novel P. Syringae phage (L17 and S8) onto KB plates. We also attended a lunch put on my MSU GRIT. The graduate students involved talked about the application process for graduate school and answered any questions that the undergraduates had about the process. The team found it very helpful and informational.

##### July 26th: EOP's and Lysate Confirmation

Both the EOP's and spot plating from yesterday were successful. We have high titer lysates for each environmental phage. We also made 30mL culture of E. coli and infected it with T7. We left this in the shaker and waited for the samples to crash. After crashing, we then filtered the samples and stored them in the 4 degrees Celsius refrigerator. We also reviewed the MIDSURE poster and got our poster printed.

##### July 29th: CRYO-EM Facility

Jenna, Roxy, and Anna went to the CRYO-EM facility here on MSU campus with Dr. Subramanian to do negative staining imaging. Before we can do anything with the CRYO-EM imaging, we had to check to see how our high titer lysates were. This process is called negative staining and we were able to see the process of how to glow discharge a copper disk and put our lysate samples on the disk.

![](https://static.igem.wiki/teams/4135/wiki/igem-notebook/img-4478.jpg)

This is the negative staining part with uracil salts on the copper disks.

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![](https://static.igem.wiki/teams/4135/wiki/igem-notebook/img-5894.png)

This is the glow discharging part. This helps the surface stay even and help the sample stick.

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![](https://static.igem.wiki/teams/4135/wiki/igem-notebook/img-5895.png)

Dr. Subramamian is showing us the ThermoFisher Talos Arctica CRYO-EM microscope.

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##### August 1st-August 2nd: DNA Sequences and Wiki

Today we had a meeting with Sundhar about the DNA sequences. We are preparing our own to order through IDT by tomorrow. The plans with John Dover for tomorrow are going to be delayed hopefully to the end of this week because next week, he is having surgery and will not be in to help us. We need to make a higher titer lysate to do genome sequencing with John Dover. We will contact him once we have our higher titer set. We did a lot of wiki stuff today and we will continue tomorrow. We are also making an E.coli overnight in LB to have for the crashing tomorrow.
Tomorrow we are planning to meet at the STEM building at 10:30am do crash the titers we have. While the titers are crashing, we will be doing wiki and DNA sequencing stuff. We akso have a meeting with the Tec-Chihuahua team to talk and ask questions about the wiki page.

##### August 12th: Calibration Kit

Today we are looking at the distribution kits that iGEM sent out to us. We are searching for E. coli DH5 alpha competent cells for these experiments to work. All the experiments will take a total time of 3 days. We are measuring the fluorescence and the OD of all the samples and transformed samples we have. Everyone on the team should look over the protocols and the data sheets to get familiar with the experiments by Monday. On Sunday, someone will come in and make an overnight of the E. coli DH5 alpha in LB + Cam ~5-10mL worth.

##### August 15th: Calibration Kit

Today we are looking at the distribution kits that iGEM sent out to us. We are searching for E. coli DH5 alpha competent cells for these experiments to work. All the experiments will take a total time of 3 days. We are measuring the fluorescence and the OD of all the samples and transformed samples we have. Everyone on the team should look over the protocols and the data sheets to get familiar with the experiments by Monday. On Sunday, someone will come in and make an overnight of the E. coli DH5 alpha in LB + Cam ~5-10mL worth.

##### August 16th: Genome Sequencing

We finally got to do genome sequencing with John Dover. We were given the protocol and John Dover was supervising us while we were conducting the experiment in his lab.

![](https://static.igem.wiki/teams/4135/wiki/igem-notebook/img-4910.jpg)

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![](https://static.igem.wiki/teams/4135/wiki/igem-notebook/68234786984-251406fb-395a-415b-988f-da0dd0fcf8d7.jpg)

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##### August 18th: Gibson and GoTaq

A vector of 4:1 of the Gly279 and Val344 into pGRB was inserted via Gibson Cloning Protocol, Product was dialyzed t for 15min in DI water and mb, electroporated it into DH10B cells, and plated 100uL and 400uL onto LB+Carb plates.

The 100uL plates of each, Val344 & Gly279, will be used for GoTaq PCR (Colony PCR) SOP. 6 colonies from each plate were chosen. The control will be an uncut pGRB vector for comparison, no positive control as there was no plasmid with any inserts was available. Ran the GoTaq at the usual setting with 30s for the elongation time at 72c since ~300bp is expected.

the plates with colonies are seen here:

![](https://static.igem.wiki/teams/4135/wiki/igem-notebook/getimage-1.png)

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pGRB check primers SP039 and SPK040 from Sundhar were used for checking if the pGRB Gibson clones were successful or not.
The two check primers without any insert will yield 138bp from SPK039 to SPK040.

This was done another round for extra possible colonies that may have the insert. The following are gel images of the colonies picked and screened using GoTaq colony PCR as described above, Circled colonies were colony C and Triangle colonies were colony T:

![](https://static.igem.wiki/teams/4135/wiki/igem-notebook/getimage-2.png)

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![](https://static.igem.wiki/teams/4135/wiki/igem-notebook/getimage-3.png)

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The T3 colony was then picked for an overnight and frozen stock to be miniprepped the next day.

##### August 19th: Miniprep

T3 Val344-pGRB vector overnight was used for a frozen stock, with the rest being used for a miniprep. The miniprep yielded 344.5ng/uL which will be used to electroporate into BW cells to conjugate into DH10B cells with the pRedCas9 plasmid.

##### September 7th: Electroporation

Electroporated Val344-pGRB gRNA (T3 colony DNA) vector in electrocompetent E. coli BW cells and recovered in 500uL of SOC with 5uL of DAP for 1hr. Following recovery, 400uL and 100uL were plated separately on LB+Carb plates (+100uL DAP) and incubated overnight at 37c.

##### CRISPY-BRED/BRIP

Gene modification technique utilizing gene editing tool CRISPR and recombination machinery lamda-red for bacteriophage. The following technique was created for model bacterium E. coli and coli phage but can be adapted for other organisms using respective recombination, CRISPR, and transformation of the new organisms.
Following procedure was adapted from Graham Hatfull Lab and Xueming Zhao labs..

###### Plasmid Information

pGRB plasmid with empty backbone for cloning and expression of crRNA in E. coli Addgene database #71539
pRedCas9 plasmid for constitutive expression of Cas9 system and IPTG inducible expression of Lamda-Red recombineering system and plasmid curing system for CRISPR editing in E. coli.
Addgene database #71541

###### Guide RNA Information

To construct a working CRISPR system, a crRNA is needed to guide the CRISPR system. The crRNA will be 20nt long and must be on the coding strand of the DNA with a PAM sequence (5'-NGG-3' repeat) downstream of the gene of interest.
The crRNA template will be placed in the pGRB plasmid using Gibson cloning or similar techniques creating the guide RNA (gRNA) system.

![](https://static.igem.wiki/teams/4135/wiki/igem-notebook/getimage-4.png)

Image 1. 20nt crRNA highlighted in green flanked with a J23119 promoter, gRNA scaffold and another J23119 Homology end for Gibson cloning into pGRB vector. Sequence in green is interchangeable depending on target, this is the crRNA.

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![](https://static.igem.wiki/teams/4135/wiki/igem-notebook/getimage-5.png)

Image 2. T7 genome with the C-terminus crRNA 20nt region highlighted in cyan representing the sequence mentioned in Image 1. Downstream is the PAM sequence 'tgg' on the coding strand highlighted in blue.

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![](https://static.igem.wiki/teams/4135/wiki/igem-notebook/getimage-6.png)

Image 3. Homologous substrate of the C-terminus region with the addition of our SpyTag protein and linker added to the C-terminus region. Each homology arm was 100nt upstream and downstream in length for a total of 200nt excluding the linker-SpyTag addition.

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Prior to following experiments we ordered the gRNA as seen in Image 1 with the gRNA scaffold and pGRB plasmid promoter homology as an dsDNA oligo sequence from IDT. Our crRNA was chosen with the NGG repeat on the coding strain seen in image 2. We also ordered the homologous substrate seen in Image 3 as a dsDNA oligo sequence also from IDT.

###### Creating Plasmids: Digested the pGRB vector using Spe1 restriction enzyme with the following reaction mixture: - 1 µL Spe1\_HF - 1 µL CIP - 5 µL 10x Cutsmart - 1000ng pGRB - 22.8µL Nuclease free H2O Incubated at 37˚C for 2 hours then held at 4˚C 2-way 10µL Gibson reaction was done using the following reaction mixture: - 1.7µL pGRB Spe1 digest - 3.3µL gRNA C-term oligo - 5µL 2x Gibson MM Incubated at 50˚C for 1 hour. Resulting Gibson reaction was dialyzed and placed into -20˚C for storage. Creating Strains: Dialyzed pGRB\_Cterm plasmid was electroporated into electrocompetent E. coli DH10B, recovered in rich SOC then plated onto LB+Amp plated grown at 37˚C. Colonies were picked and screened using GoTaq colony PCR using primers 50nt up and downstream of the Spe1 cut site, a colony with the 161nt insertion was picked reverified and a frozen stock was made. Mating strain E. coli BW25113 was electroporated with the pRedCas9, recovered in rich SOC media for 1 hour before being plated on LB+DAP+Spec and grown at 30˚C. pRedCas9 is temperature sensitive . The next day a frozen stock was made using a single isolated colony. Both mating strain BW25113+pRedCas9 and DH10B+pGRB\_Cterm was spotted on top of one another on LB+DAP and grown overnight at 30˚C. Next day, the mixture growth was streak plated on LB+Spec+Carb selecting for DH10B+pRedCas9+pGRB\_Cterm unselecting for the mating strain BW25113 that required DAP to grow. Single colony of the E. coli DH10B pRedCas9+pGRB\_Cterm streak plate was picked and made into a frozen stock and an overnight liquid stock. Making Electrocompetent Cells: Grow E. coli DH10B pRedCas9+pGRB\_Cterm cells from an overnight culture until OD600 = 0.35-0.40. Spin down 2ml of culture in microcentrifuge for 1 minute at full speed at room temperature. Remove supernatant. Resuspend pellet in 2ml of ice cold sterile H2O. Centrifuge for 1 minute at full speed at 4 degrees C. At this point, keep all cells on ice. Remove supernatant. Resuspend pellet in 1ml of ice cold sterile H2O. Centrifuge for 1 minute at full speed. Remove supernatant. Resuspend pellet in 100ul of 10% glycerol. Centrifuge for 1 minute at full speed. Remove supernatant. Resuspend pellet in 40ul of 10% glycerol. Make 20uL aliquots of cells in PCR strip tubes. CRISPY-BRED: Plate 8mL of MMB agar with 250uL of E. coli DH10B pRedCas9+pGRB\_Cterm cells from an overnight and pour onto small petri dishes. Let cool for 10mins. Electroporate 1uL of dsDNA substrate (Image 3 sequence) with 1uL of phage DNA into electrocompetent E. coli DH10B pRedCas9+pGRB\_Cterm cells. Let cells recover and grow in 500uL SOC for 28mins for one phage replication cycle. Plate cells with 5mL-8mL of MMB agar and recovered cells as top agar onto previously made plates Grow plates overnight at 30˚C. Next day, pick and use GoTaq colony PCR to screen phage with primers flanking homology arms to detect insert of sequence. CRISPY-BRIP This method is used for phage with genome sized too large to electroporate into cells (>40-50kb) Plate 8mL of MMB agar with 250uL of E. coli DH10B pRedCas9+pGRB\_Cterm cells from an overnight and pour onto small petri dishes. Let cool for 10mins. Electroporate 1uL of dsDNA substrate (Image 3 sequence) ] into electrocompetent E. coli DH10B pRedCas9+pGRB\_Cterm cells. Let cells recover and grow in 500uL SOC with the addition of phage for an MOI of 1-2. Plate cells with 5mL-8mL of MMB agar and recovered cells as top agar onto previously made plates Grow plates overnight at 30˚C. Next day, pick and use GoTaq colony PCR to screen phage with primers flanking homology arms to detect insert of sequence.

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{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Design. Build. Test. Learn. Repeat {% endblock %}
{% block page\_content %}

#### Design: Round 1

###### Phage: what we knew

* Phage are very specific to their individual host ensuring no broad-host microbiome problems.
* Ubiquitous and natural bacteria killers making them cheap and easy to use.
* Useful against drug-resistant pathogens.

###### Understanding the problems

* Phage are less efficient than other biocontrols like the aforementioned pesticide or antibiotics leading to poor robustness.
* Phage specificity leads to the need of specialized cocktails for complex infections, especially in human phage therapeutics.
* Uncertainty in the type of phage and possibility of lysogenic phage reduce the viability of possible therapeutics.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %} The Protocols and Methods That Got Us Where We Are.{% endblock %}
{% block page\_content %}

* [King's Broth Preparation](#1)* [Phage Isolation](#3)* [Colony Forming Unit Test](#5)* [dsDNA Phage Genome Extraction Protocol](#9)

* [Filtrate Creation](#2)* [Effiency of Plaquing Test](#4)* [High Titer Lysate Creation](#6)* [Crispy BRip Method](#8)

## King's Broth Preparation

---

So many experiments in microbiology require the creation of media that it is often forgotten in terms of procedures. Kings Broth is an agar and broth recipe important of the growth of Pseudomonas strains, our model pathogen, and the name sake of our team, the Phage Court.

### Materials per Liter

* 1L Distilled Water* 20g Difco Proteose Peptone #3* 1.5g K2HPO4* 1.5g MgSO4•7H2O* 10mL Glycerol* 15g Agar (if making solid media)

            ---

            ### Instructions

            1. Bring the water to a boil.

            2. Add in the peptone, and stir until dissolved.

            3. Add the other ingredients, and stir until dissolved before cutting the heat.

            4. Bottle, and autoclave to sterilize.

## Filtrate Creation

---

Phage exist almost everywhere in nature, and anywhere your host may be found it is likely there is also phage to target it. Our first attempt at this process used environmental water and leaf samples did not yield phage. Only on our second attempt when we included soil samples did we produce viable results.

### Materials per Sample

* 2 50mL Falcon Tube* 30mL distilled Water* 1m of 500mM CaCl2* 10mL Kings Broth* 1mL of Overnight Liquid Host Culture

          ---

          ### Instructions

          1. Add the distilled water to the first falcon tube, and fill the rest with soil from the host's preferred environment.

          2. Shake the mixture before letting it settle.

          3. In the second tube, prepare the rest of the ingredients, and add 10mL of water from tube A.

          4. Incubate the mixture at 28°C for 24 hours.

## Phage Isolation

---

Because many different phage may be contained in one sample, this procedure allows us to purify each individual phage based on host focus.

### Materials per Sample

* 18mL of KB agar* 200mL 500mM CaCl2* 100 mL overnight host* Roughly 4 mL of Phage Buffer* 10μL of filtrate* One large plate

            ---

            ### Instructions

            1. Section the large plate into 6 sections.

            2. While the agar is liquid, add the overnight culture alongside the calcium chloride and pour the plate.

            3. Set up a row of 5 microtubes, and add 90μL of phage buffer to each.

            4. Perform serial dilutions by first adding 10μL of filtrate to the first and then taking 10 from there for the next etc.

            5. Once the plate has solidified, add 10μL of each dilution, and the original filtrate thrice in each secion, one for each row.

            6. Incubate the plates at 28°C checking for plaques each day.

            7. Once individual plaques have formed, pipette the agar of the plague up with a serological pipette, and put it in 100mL of overnight before incubating it for 24 hours at 28°C, this is your now lysate.

            8. Repeat this process with the lysate until the plaques all look the same.

## Efficiency of Plaquing Test

---

This process is very similar to isolating phage, but takes place with purified high titer lysate to determine how many infecteous phage are in it per Liter.

### Materials per Sample

* 18mL of KB agar* 200mL 500mM CaCl2* 100mL overnight host* Roughly 4 mL of Phage Buffer* 10μL of lysate* One large plate

            ---

            ### Instructions

            1. Section the large plate into 3 rows of 12.

            2. While the agar is liquid, add the overnight culture alongside the calcium chloride and pour the plate.

            3. Set up a row of 12 microtubes, and add 90μL of phage buffer to each.

            4. Perform serial dilutions by first adding 10μL of filtrate to the first and then taking 10 from there for the next ect.

            5. Once the plate has solidified, add 5μL of each dilution thrice, one for each row.

            6. Incubate the plates at 28°C for the ammount of time previously determined for plaques to form.

            7. Count the plaques at the dilution where you are first able to see individual plaques and use this to calculate your titer.

            8. Repeat this process three times with the same lysate for accurate data.

## Colony Forming Unit Test

---

Colony forming unit tests or CFUs can allow us to determine the amount of bacteria relative to the optical density of a sample.

### Materials per Sample

* 108mL of KB agar* Roughly 4 mL of King's Broth* 2mL of overnight host* 1 mL distilled water* 6 large plates* Two Cuevettes* An OD reader

              ---

              ### Instructions

              1. Section the large plate into 3 rows of 12.

              2. Pour the plates with 18 mL KB agar each

              3. Set up 5 2.5mL microtubes each with 1mL of KB broth

              4. Perform serial half dilutions of the overnight host into the 5 microtubes.

              5. Calibrate the OD reader with a cuvette with 1 mL of DI water, before measuring first the overnight, then each half dilution all in the same cuvette.

              6. Set up a row of 12 microtubes, and add 90μL of kings broth to each.

              7. Perform serial dilutions with 10 mL of the overnight.

              8. Repeat 6 and 7 with each of the half dilutions.

              9. Add 5μL of each 10th dilution thrice, one for each row.

              10. Repeat 9 using each half dilution's set of 12 each on a seperate labeled plate

              6. Incubate the plates at 28°C for the amount of time previously determined for host colonies to form.

              7. Count the colonies at the dilution where you are first able to see individual colonies on each plate and use this to calculate your titer at each half dilution.

              8. Plot the colony forming units against the opical density to create a curve on which to predict CFU from OD.

## High Titer Lysate Creation

---

To get lots of DNA for DNA Prep, you first need a lot of phage from which to get the DNA. This procedure is meant to create a solution containing many phage per mL, usually the target being around 10^9.

### Materials per Liter

* 30mL Kings Broth* 100μL Lysate* Plate of Host Culture

      ---

      ### Instructions

      1. Inoculate the broth with host, and place in a shaking incubator.

      2. Monitor the broth until cloudy, after which the phage should be immediately added.

      3. Continue incubating until the broth clears again.

      4. Refrigerate the lysate and perform an EOP to determine titer.

## Crispy BRIP Method

---

Gene modification technique utilizing gene editing tool CRISPR and recombination machinery lamda-red for bacteriophage. The following technique was created for model bacterium E. coli and coli phage but can be adapted for other organisms using respective recombination, CRISPR, and transformation of the new organisms. This is our most complex procedure and will be broken into pieces.

### Instructions

1. To construct a working CRISPR system, a crRNA is needed to guide the CRISPR system. The crRNA will be 20nt long and must be on the coding strand of the DNA with a PAM sequence (5'-NGG-3' repeat) downstream of the gene of interest

2. The crRNA template is then placed in the pGRB plasmid using Gibson cloning or similar techniques creating the guide RNA (gRNA) system

### 3. Creating Plasmids:

4. Digest the pGRB vector using Spe1 restriction enzyme with the following reaction mixture:

* 1 µL Spe1\_HF* 1 µL CIP* 5 µL 10x Cutsmart* 1000ng pGRB* 22.8µL Nuclease free H2O

          5. Incubate at 37˚C for 2 hours then held at 4˚C

          6. 2-way 10µL Gibson reaction is done using the following reaction mixture:

          * 1.7µL pGRB Spe1 digest* 3.3µL gRNA C-term oligo* 5µL 2x Gibson MM

                7. Incubate at 50˚C for 1 hour.

                8. Dialyze and place into -20˚C for storage.

                ### Creating Strains:

                9. Electroporate the dialyzed pGRB\_Cterm plasmid was into the electrocompetent E. coli DH10B, recovere in rich SOC then plate onto LB+Amp grown at 37˚C.

                10. Picked and screen colonies using GoTaq colony PCR using primers 50nt up and downstream of the Spe1 cut site, pick reverify and create a frozen stock from a colony with the 161nt insertion.

                11. Electroporate the mating strain E. coli BW25113 with the pRedCas9, recovere in rich SOC media for 1 hour before plating on LB+DAP+Spec at 30˚C.

                12. Make a frozen stock using a single isolated colony.

                13. Both mating strain BW25113+pRedCas9 and DH10B+pGRB\_Cterm need to be spotted on top of one another on LB+DAP and grown overnight at 30˚C.

                14. Next day, the mixture growth was streak plated on LB+Spec+Carb selecting for DH10B+pRedCas9+pGRB\_Cterm unselecting for the mating strain BW25113 that required DAP to grow.

                15. Pick a single colony of the E. coli DH10B pRedCas9+pGRB\_Cterm streak plate and make into a frozen stock and an overnight liquid stock.

                ### Making Electrocompetent Cells:

                16. Grow E. coli DH10B pRedCas9+pGRB\_Cterm cells from an overnight culture until OD600 = 0.35-0.40.

                17. Spin down 2ml of culture in microcentrifuge for 1 minute at full speed at room temperature.

                18. Remove supernatant. Resuspend pellet in 2ml of ice cold sterile H2O.

                19. Centrifuge for 1 minute at full speed at 4 degrees C. At this point, keep all cells on ice.

                20. Remove supernatant. Resuspend pellet in 1ml of ice cold sterile H2O.

                21. Centrifuge for 1 minute at full speed.

                22. Remove supernatant. Resuspend pellet in 100ul of 10% glycerol.

                23. Centrifuge for 1 minute at full speed.

                24. Remove supernatant. Resuspend pellet in 40ul of 10% glycerol.

                25. Make 20uL aliquots of cells in PCR strip tubes.

                ### CRISPY-BRIP

                26. Plate 8mL of MMB agar with 250uL of E. coli DH10B pRedCas9+pGRB\_Cterm cells from an overnight and pour onto small petri dishes. Let cool for 10mins.

                27. Electroporate 1uL of dsDNA substrate (Image 3 sequence) ] into electrocompetent E. coli DH10B pRedCas9+pGRB\_Cterm cells.

                28. Let cells recover and grow in 500uL SOC with the addition of phage for an MOI of 1-2.

                29. Plate cells with 5mL-8mL of MMB agar and recovered cells as top agar onto previously made plates.

                30. Grow plates overnight at 30˚C.

                Next day, pick and use GoTaq colony PCR to screen phage with primers flanking homology arms to detect insert of sequence.

## dsDNA Phage Genome Extraction Protocol

---

This protocol has been adapted from a procedure from the lab of Dr. Kristen Parent.

### Materials per Experiment

* High Titer Phage Lysate* 100 mg/mL Lysosome* 10 mg/mL Rnase A* DNase 1 (NEB, M0303S)* 10x DNase 1 Buffer* 10% SDS* 10 mg/mL proteinase K* Phenol:Chloroform:Isoamyl Alcohol (25:24:1)* Chloroform* 100% EtOH* 3 M Sodium Acetate* 70% EtOH

                        ---

                        ### Instructions

                        1. In a 2 mL microcetrifuge tube, add the following:

                        + 738 μL of high titer phage lysate+ 9μL of 100 mg/mL Lysozyme (1 mg/mL final)+ 9μL 10 mg/mL RNase A without EDTA+ 9μL of NEB DNase 1+ 90 μLof 10x DNase 1 Buffer

                        2. Mix by inversion or pulse vortex, and incubate at 37°C for 90 minutes.

                        3. Inactivate the DNase by incubating at 75°C for 10 minutes.

                        4. Let the sample cool to room temp.

                        5. Add 10μL of 10mg/mL Proteinase K and 50 μL of 10% SDS, and mix by inversion.

                        6. Incubate at 55°C for 1 hour.

                        7. Let the sample cool.

                        8. Add 960μL of Phenol:Chloroform and mix by inverting several times until the sample is cloudy.

                        9. Centrifuge at 12,000 RPM for 10 minutes at room temperature.

                        10. Transfer the aqueous layer to a new 2 mL tube (be careful not to aspirate the organic layer).

                        11. Add an equal volume of chloroform (likely 750 mL) and mix by inverting the tube several times. Using chloroform rather than phenol:chloroform helps remove phenol which can shift the peaks in the spectrophotometer.

                        12. Centrifuge at 12,000 RPM for 10 minutes at room temperature.

                        13. Transfer the aqueous layer to a to a new 2 mL tube (roughly 550μL).

                        14. Add two volumes of 100% ethanol and then 1/10 volume of 3 M Sodium Acetate. Mix by inversion.

                        15. Incubate at -20°C for roughly 60 minutes. You can leave this overnight if you wish.

                        16. Centrifuge at 14,000 RPM for 5-10 minutes at room temperature.

                        17. Carefully decant the supernatant into a waste container.

                        18. Add 500μL of 70% Ethanol, being careful not to disturb the pellet. Carefully invert the tube once or twice.

                        19. Centrifuge at 14,000 RPM (or max speed) for 2-3 minutes at room temperature.

                        20. Carefully decant the supernatant into a waste container, and pipette off any ethanol that remains.

                        21. Air dry the pellet for for 15 minutes or until no droplets. Do not overdry the pellet or it will be difficult to dissolve.

                        22. Resuspend the pellet in 50-100μL of DI-H2O or a sufficient volume to dissolve the pellet.

                        23. Incubate at room temperature for 30 minutes or until it dissolves.

                        24. Check the DNA concentration in the spectrophotometer, and store the remaining DNA at -20°C.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

## Aims of Research

---

##### Aim One: Isolation and Purification of Environmental Phage

* Isolate environmental phage against P. syringae DC3000 pv. from water samples. ​
* Identify plaquing efficacy of environmental phage on host.

##### Aim Two: Modify Phage Capsids for Increased UV-C resistance

- Model commercial P. syringae phage capsids and identify modification sites for SpyTag addition. ​
- Use the BRED technique to add SpyTag peptide to the capsid.​
- Test mutant phage susceptibility to UV-C exposure. ​​

## Results

---

###### Aim one: P. syringe phage, isolated from Red Cedar and Tomato soil, presented two distinct morphologies:​ Figure 1 demonstrates large plaques from phage L17 and small plaques from phage S8​

![](https://static.igem.wiki/teams/4135/wiki/screen-shot-2022-08-16-at-10-41-33-am.png)

Figure 1: Efficiency of plaquing (EOP) of environmentally isolated phage on P. Syringae DC3000 on KB agar. Panel A demonstrates phage S8 with a titer of 5.12E9; panel B demonstrates phage L17 with a titer of 1.07E10 on a P. syringae DC3000 lawn.

+ add in phage images
+ Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://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

---

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. 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.

## Inspirations

---

* [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)

{% endblock %}