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!
- 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
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.
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.
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.
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.
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.
Figure 1. James Suggitt did his EOPs in triplicates replicating all the dilutions in a horizontal manner.
Figure 2. Anna Kim did her EOPs in triplicates replicating all the dilutions by dividing up the plate into sections.
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.
Figure 1. CFU plate 1
Figure 2. CFU plate 2
Figure 3. CFU plate 3
Figure 4. CFU plate 4
Figure 5. CFU plate 5
Figure 6. CFU plate 6
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:
Figure 7. Data from Growth Curve Calculations
Figure 8. Graph from Growth Curve Calculations
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.
This is the negative staining part with uracil salts on the copper disks.
This is the glow discharging part. This helps the surface stay even and help the sample stick.
Dr. Subramamian is showing us the ThermoFisher Talos Arctica CRYO-EM microscope.
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.
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:
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:
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..
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
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.
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.
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.
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.
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.
Digested the pGRB vector using Spe1 restriction enzyme with the following reaction mixture:
Incubated at 37˚C for 2 hours then held at 4˚C
2-way 10µL Gibson reaction was done using the following reaction mixture:
Incubated at 50˚C for 1 hour. Resulting Gibson reaction was dialyzed and placed into -20˚C for storage.
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.
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.
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.
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.