Team:UCSC/Notebook
Dry Lab
During the first week of the project, we mainly focused on developing a plan for the outreach section based on the medal requirements on the iGEM website. We looked for companies and nonprofits that would be able to share information on the issues with us, as well as doctors who worked in the areas most affected by Newcastle disease. We also set up meetings with people on campus to better help us develop our plans. Work on the wiki began with general formatting based on examples from previous years teams.
Significant Events:
Contacted Tyson Foods, Perdue, and Foster Farms on Facebook:
Participants: Nikki
Notes: Contacted for funding and for general vaccination practices
Results: Tyson and Perdue responded with information that they give to farmers on vaccination as well as grant forms.
Contacted Dr. Phil Berman:
Participants: Melody
Notes: Contacted him to find out about developing vaccines
Results: Meeting set up for July 1 to discuss vaccines
Contacted Priya of the Silver Lab:
Participants: Jonah
Notes: Contacted the Silver Lab at Harvard who we had been in touch previously
Results: All of the plasmids that the Silver Lab had worked on were sent to us.
Contacted the Pielak Lab:
Participants: Iris
Notes: Contacted the Pielak Lab at the University of North Carolina at Chapel Hill who had performed previous work on IDP’s.
Results: Received a plasmid for CAHSD in July.
Meeting with Susan Strome, Chair of MCD Bio:
Participants: Marcella, Shayan, Nikki, Preet, Aren, Rita
Notes: N/A
Results: Presented our project and iGEM to the MCD chair and were given funding at a later date.
Participants: Melody
Notes: Contacted him to find out about developing vaccines
Results: Meeting set up for July 1 to discuss vaccines
Contacted Priya of the Silver Lab:
Participants: Jonah
Notes: Contacted the Silver Lab at Harvard who we had been in touch previously
Results: All of the plasmids that the Silver Lab had worked on were sent to us.
Contacted the Pielak Lab:
Participants: Iris
Notes: Contacted the Pielak Lab at the University of North Carolina at Chapel Hill who had performed previous work on IDP’s.
Results: Received a plasmid for CAHSD in July.
Meeting with Susan Strome, Chair of MCD Bio:
Participants: Marcella, Shayan, Nikki, Preet, Aren, Rita
Notes: N/A
Results: Presented our project and iGEM to the MCD chair and were given funding at a later date.
Participants: Iris
Notes: Contacted the Pielak Lab at the University of North Carolina at Chapel Hill who had performed previous work on IDP’s.
Results: Received a plasmid for CAHSD in July.
Meeting with Susan Strome, Chair of MCD Bio:
Participants: Marcella, Shayan, Nikki, Preet, Aren, Rita
Notes: N/A
Results: Presented our project and iGEM to the MCD chair and were given funding at a later date.
Wet Lab
The first week consisted of reading papers and determining which experiments we would need to do to complete the project. After determining a general outline of what experiments we would be performing, a reagents list was created based on estimates of how much of each reagent we would need. We then began purchasing the reagents that we would need for the first experiments. We also organized the lab space for future use, acquired proper PPE, and were trained on proper pipette technique and dishwasher use.
Important Tasks
Dishwasher Training
Participants: Everyone
Protocols: N/A
Notes: N/A
Results: Everyone is now dishwasher trained
Pipette Training and Calibration
Protocols: N/A
Notes: N/A
Results: Proper pipette technique reviewed and pipettes calibrated.
Experimental Outline Meeting, Reagent Sheets and Protocols
Participants: James, Preet, Jonah, Iris, Andrew, Ike
Protocols: Gathering them.
Notes: N/A
Results: Determined a rough outline for the experiments ,created Google Sheets of reagents we may need with estimates on amounts, costs, and uses. We then gathered specific protocols, from online and book sources for the experiments.
Dry Lab
This week additional time was spent to further develop our plans for outreach. We focused more on developing the story of our project and following up on responses from the first week. We also looked for people who had experience in resource constrained countries to see if our project would be of use to them. Crowdfunding officially started and so members of the team reached out to friends and family for possible donations. We also looked towards large companies, grants, and departments on campus.
Significant Events:
Meeting with Dr. Philip Berman
Participants: Melody, James, Rita, Shayan
Notes: N/A
Results: Learned about the difficulties of vaccine development, the cold chain, and the implications of our project. Learned new terminology.
Contacted Novartis, Boehringer Ingelheim, and MERCK for General Vaccine information and Storage Guidelines
Participants: Nikki
Notes: N/A
Results: Gave us general storage guidelines and told us that our project would be useful to them.
Outreach to Worldbuilders
Participants: Varuna, Ike
Notes: N/A
Results: Trying to contact them to form a relationship with their partner, Heifer.
Emailed Dr. Arthur Dover
Participants: Marcella
Notes: N/A
Results: Crowdfunding began later this week and we raised a significant portion of our total funding through it, over the next 3 months.
Interview Questionnaire Draft
Participants: Rita, Martina, Melody
Notes: N/A
Results: Created a questionnaire for future interviews.
Participants: Nikki
Notes: N/A
Results: Gave us general storage guidelines and told us that our project would be useful to them.
Outreach to Worldbuilders
Participants: Varuna, Ike
Notes: N/A
Results: Trying to contact them to form a relationship with their partner, Heifer.
Emailed Dr. Arthur Dover
Participants: Marcella
Notes: N/A
Results: Crowdfunding began later this week and we raised a significant portion of our total funding through it, over the next 3 months.
Interview Questionnaire Draft
Participants: Rita, Martina, Melody
Notes: N/A
Results: Created a questionnaire for future interviews.
Participants: Marcella
Notes: N/A
Results: Crowdfunding began later this week and we raised a significant portion of our total funding through it, over the next 3 months.
Interview Questionnaire Draft
Participants: Rita, Martina, Melody
Notes: N/A
Results: Created a questionnaire for future interviews.
Wet Lab
This week we spent more time finalizing our experiments. In addition, the team was trained on using the autoclave and biohazard disposal. To verify our protocols were correct a small subsection of the team performed a trial run of one of the plasmids sent to us by the Silver Lab which had arrived earlier that week. This test run included transformation, index plating, liquid cultures, and finally miniprepping the plasmid. We also learned how to make kanamycin stocks, LB plates, and developed a labeling method for our samples.
Important Tasks
Autoclave Training
Participants: Iris, James, Andrew, Preet, Jonah, Liana
Protocols: N/A
Notes: N/A
Results: Members of the team were trained on how to safely use the autoclave
Solubilization of CAHS1(G1) off of Blotting Paper & Transformation (Test):
Participants: Iris, Jonah, Preet, Andrew
Protocols: Plasmid Solubilization Transformation
Notes: A plasmid we did not intend to use (non e. Coli optimized) was solubilized off of blotting paper and transformed into DH5a e. coli.
Results: We were able to confirm that we could solubilize plasmids off of the paper and use it for transformation.
Index Plating
Participants: Iris, Jonah, Preet
Protocols: Index Plating
Notes: We plated 9 separate colonies onto index plates.
Results: We were able to grow these isolated colonies as separate indices to test for successful transformations.
Liquid Broth Inoculation Test
Participants: Iris, Jonah, Preet
Protocols: Liquid Inoculation(Test)
Notes: In order to test for the successful transformation of our vector into the colonies, we made liquid cultures that we could then miniprep.
Results: In order to test for the successful transformation of our vector into the colonies, we made liquid cultures that we could then miniprep.
Dry Lab
We met with Alexis Morgan, social media/outreach head at the genomics institute to gain insight how we should develop our brand and reach out to people. We also met with Rodger Trippel for advice on fundraising. We set up a meeting with Dean Koch of PBSCI. We created abstract graphics were created for the description page. We also began researching the ethical implications associated with our project. We also had a phone call with Thomas Boothby whose previous research on IDP’s had spurred us into our project.
Significant Events:
Meeting With Thomas Boothby
Participants: Melody, Marcella, Martina, Jonah, Preet, Shayan, Marcus, Martina, Nikki, Aren, Rita
Notes: N/A
Results: We explained our project to him and he told us about growing up in Kenya and how important heat- stable vaccines would be there. We also discussed possible collaborations in the future.
WorldBuilders Response
Participants: Varuna
Notes: N/A
Results: They gave us a contact for the director of philanthropy at Heifer International. We contacted them and set up a meeting later in the month.
Meeting with Bari Nazario:
Participants: Jonah, Aren, Preet
Notes: N/A
Results: Bari gave us filters, vacuum aspirators, biohazard waste containers, and instructions on sterilization of the BSL2 room.
Participants: Varuna
Notes: N/A
Results: They gave us a contact for the director of philanthropy at Heifer International. We contacted them and set up a meeting later in the month.
Meeting with Bari Nazario:
Participants: Jonah, Aren, Preet
Notes: N/A
Results: Bari gave us filters, vacuum aspirators, biohazard waste containers, and instructions on sterilization of the BSL2 room.
Wet Lab
After finishing the test run of CAHS1, we began dividing up the team into groups of two to three to begin work on individual plasmids. Just like the test run, these included transformation, index plates, liquid cultures, and purification. Later, we adjusted our methods and added in colony PCR before making liquid cultures, so we could get more immediate results by running our PCR products on a gel. This allowed us to verify if what we had transformed was in fact the correct plasmid based on the length of the band we saw on the gel. A reagents lists was made for the future experiments that would take place in the BSL2 room. The necessary equipment was then moved into the room and then the room and equipment were sterilized. An additional plasmid (CAHSD) was received from the Pielak Lab and was transformed.
Important Tasks
Miniprep of CAHS 1 (G1) (Test)
Participants: James, Preet, Iris, Andrew, Jonah
Protocols: Miniprep , Transformation
Notes: We ran the liquid cultures using the Zymo Miniprep kit. This would allow us to verify the production of our plasmid in our transformed colonies.
Gel Electrophoresis of CAHS 1 (G1) Samples (Test)
Participants: Andrew, Aren, James
Protocols: Gel Electrophoresis Transformation
Notes: By running the plasmids on an agarose gel, we are able to check if the correct plasmid is being produced by comparing its length against a DNA ladder.
Results: We were able to confirm that most of our colonies were transformed correctly and contained the plasmid.
Solubilzation and Transformation of E. coli optimized CAHS 1 (D4)
Participants: Liana, Andrew
Protocols: Transformation
Notes: E. coli optimized CAHS 1 was transformed into DH5a E. coli. Transformation was done by directly adding blotting paper to transformation tube
Results: Failed Transformation. Blamed on lack of plasmid transfer form blotting paper to surrounding cells.
Solubilzation and Transformation of CAHS 2 (D3) & CAHS 7 (A2)
Participants: Varuna, Nikki, Martina
Protocols: Plasmid Solubilization , Transformation
Notes: Plasmids were transformed into DH5a E. coli
Results: Growth was seen on the transformation plates.
Solubilization and Transformation of rvLEAM (H4)
Participants: Melody, Marcella, Rita
Protocols: Plasmid Solubilization, Transformation
Notes: Plasmids were transformed into DH5a E. coli
Results: Failed Transformation. Competent cells may have been left to thaw too long.
Re-Transformation of E. coli optimized CAHS 1 (D4)
Participants: Liana, Andrew
Protocols: Transformation
Notes: E. coli optimized CAHS 1 was transformed into DH5a E. coli. Transformation was done by directly adding blotting paper to transformation tube and adding 40uL of water to the paper to solubilize the plasmids into.
Results: Growth was seen on the transformation plates.
Re-Transformation of rvLEAM (H4)
Participants: Melody, Marcella
Protocols: Plasmid Solubilization , Transformation
Notes: Plasmids were transformed into DH5a E. coli.
Results: Growth was seen on the transformation plates.
Index Plating & Liquid Inoculations of CAHS 1 (D4), CAHS 2 (D3), CAHS 68135 (A2), & rvLEAM (H4)
Participants: Andrew, Liana, Melody, Marcella, Martina, Varuna, Nikki
Protocols: Index Plating & Liquid Incolulation
Notes: We plated 9 separate colonies onto index plates.
Results: We were able to grow these isolated colonies as separate indices to test for successful transformations.
Solubilization and Transformation of CAHS D
Participants: Jonah, Iris
Protocols: Plasmid Solubilization , Transformation
Notes: Plasmids were transformed into DH5a E. coli. (Plasmid from Pixie Lab)
Results: Growth was seen on the transformation plates.
Colony PCR and Gel electrophoresis of CAHS 1 (D4), CAHS 2 (D3), CAHS 68135 (A2), & rvLEAM (H4)
Participants: James, Andrew, Liana, Melody, Marcella, Martina, Varuna
Protocols: Colony PCR, Gel Electrophoresis
Notes: In order to test for the successful transformation of our vector into the colonies, we conducted colony PCR on 3 of 9 colonies using primers that would amplify the gene inserts. We ran the amplified fragments from our colony pcr on an agarose gel.
Results: Using the gel, we were able to confirm which colonies were successfully transformed with our vector.
Index Plating, Colony PCR, and Gel electrophoresis of CAHS D
Participants: Iris, Jonah
Protocols: Index Plating & Colony PCR, Gel Electrophoresis
Notes: Colony PCR failed due to incorrect primers being used.
Results: No bands were present on the agarose gels. Index plate had growth.
Sterilizing BSL2:
Participants: Iris, Jonah
Protocols: N/A
Notes: N/A
Results: Room and equipment were sterilized.
Dry Lab
Significant work was put into part one of the technical report. This report consisted of all of our research thus far and our future plans and would be submitted to our PI for review. In addition, a later version of this report would be submitted for Dean’s and Chancellor's Awards. A poster reflecting our work thus far was also developed. We remained in contact with Worldbuilders and the girls gave a presentation to Girls in Engineering about being a woman in STEM. Team and individual photos were taken and uploaded onto the website.
Significant Events:
Work On Technical Report:
Participants: James, Liana, Martina, Melody, Varuna, Rita, Marcella
Notes: N/A
Results: Significant progress was made on finishing draft one of the report.
Reached out to Thermo Fisher:
Participants: Nikki
Notes: N/A
Results: Thermo Fisher donated cell culture supplies. Thank you!
Reached out to Palo Alto Medical Foundation and travel doctor
Participants: Nikki, Marcella
Notes: N/A
Results: Offered us a tour of their lab and they stated that our project would be useful to them.
Girls in Engineering Presentation:
Participants: Liana, Martina, Melody, Varuna, Nikki, Preet, Rita, Marcella
Notes: Girls in Engineering is a program dedicated to teaching young girls about engineering and basic coding skills.
Results: Some of the women on the team talked to the girls in Girls in Engineering about their experiences in the STEM field to inspire the next generation of scientists.
Participants: Nikki
Notes: N/A
Results: Thermo Fisher donated cell culture supplies. Thank you!
Reached out to Palo Alto Medical Foundation and travel doctor
Participants: Nikki, Marcella
Notes: N/A
Results: Offered us a tour of their lab and they stated that our project would be useful to them.
Girls in Engineering Presentation:
Participants: Liana, Martina, Melody, Varuna, Nikki, Preet, Rita, Marcella
Notes: Girls in Engineering is a program dedicated to teaching young girls about engineering and basic coding skills.
Results: Some of the women on the team talked to the girls in Girls in Engineering about their experiences in the STEM field to inspire the next generation of scientists.
Participants: Liana, Martina, Melody, Varuna, Nikki, Preet, Rita, Marcella
Notes: Girls in Engineering is a program dedicated to teaching young girls about engineering and basic coding skills.
Results: Some of the women on the team talked to the girls in Girls in Engineering about their experiences in the STEM field to inspire the next generation of scientists.
Wet Lab
Training continued on transformations, minipreps, and colony PCR on plasmids that had failed at one of the aforementioned steps the week before. In addition, standard protocols were made for each of the steps. An additional plasmid was received from the Pielek Lab, and one additional plasmid was chosen from those received from the Silver Lab.
Important Tasks
Solubilization and Transformation of SAHS 10 (G3)
Participants: Liana, Andrew
Protocols: Plasmid Solubilization , Transformation
Notes: E. coli optimized SAHS 10 was transformed into DH5a E. coli.Results: Growth was seen on plate.
Index Plating, Colony PCR, and Gel electrophoresis of SAHS 10 (G3)
Participants: Andrew
Protocols: Index Plating & Colony PCR and Gel Electrophoresis
Results: All colony PCR samples showed the expected insert fragment length.
Miniprep of CAHS1 (D4), SAHS 10 (G3) & rvLEAM (H4)
Participants: Andrew, Liana, Melody, Nikki, Marcella
Protocols: Miniprep
Notes: We ran the liquid cultures using the Zymo Miniprep kit. This would allow us to verify the production of our plasmid in our transformed colonies.
Colony PCR, and Gel electrophoresis of CAHS2 (D3)
Participants: Martina, Varuna
Protocols: Colony PCR Gel Electrophoresis
Results: Gel showed major streaking. It was concluded that Colony 6 would be used for minipreps.
Gel electrophoresis of CAHS1 (D4), SAHS 10 (G3) & rvLEAM (H4)
Participants: Andrew, Melody
Protocols: Colony PCR Gel Electrophoresis
Notes: N/A
Results: Lengths of the purified plasmids were confirmed and correct.
Dry Lab
Work continued on the Technical Report, and poster. Each team member created a bio for another team member which lead to amusing results. After edits, these bios were uploaded onto the website. In addition, we were finally able to talk to Heifer directly through a phone call about our project. We also led lab tours for Girls in Engineering. Finally, we had a meeting to reschedule the deadlines we wanted to hit based on the progress we had made.
Significant Events:
Technical Report
Participants: James, Melody, Martina, Preet, Liana, Shayan, Marcella, Rita
Notes: N/A
Results: Finished draft one of the technical report and submitted it to our PI for evaluation. Everyone on the team also submitted a personal reflection along with the report.
Meeting with Suz Howell
Participants: Marcella, Varuna, Aren
Notes: N/A
Results: Met to discuss other sources of funding in the community. Suz gave us many contacts.
Meeting with Professor Matthew Sparke
Participants: Marcella
Notes: Professor in Sociology.
Results: Learned about global health perspectives on NDV in countries like Tanzania and he shared papers relevant to our project.
Heifer Meeting
Participants: Melody, Martina, Shayan, Marcella, Varuna, Rita
Notes: N/A
Results: Heifer gave us a lot of information Newcastle Disease and vaccines in resource constrained areas. We asked to partner with them and they said yes.
Meeting with Dean Wolfe
Participants: Marcella, Varuna
Notes: N/A
Results: Presented the project to the dean of Jack Baskin Engineering and received funding through the engineering department.
Outline Meeting
Participants: Ike, Varuna, Andrew, James
Notes: N/A
Results: Rescheduled our overarching goals based on our progress thus far.
Girls in Engineering Lab Tours
Participants: Liana, Aren
Notes: N/A
Results: Gave multiple lab tours to Girls in Engineering and showed them what a bioengineering lab was like.
Participants: Marcella, Varuna, Aren
Notes: N/A
Results: Met to discuss other sources of funding in the community. Suz gave us many contacts.
Meeting with Professor Matthew Sparke
Participants: Marcella
Notes: Professor in Sociology.
Results: Learned about global health perspectives on NDV in countries like Tanzania and he shared papers relevant to our project.
Heifer Meeting
Participants: Melody, Martina, Shayan, Marcella, Varuna, Rita
Notes: N/A
Results: Heifer gave us a lot of information Newcastle Disease and vaccines in resource constrained areas. We asked to partner with them and they said yes.
Meeting with Dean Wolfe
Participants: Marcella, Varuna
Notes: N/A
Results: Presented the project to the dean of Jack Baskin Engineering and received funding through the engineering department.
Outline Meeting
Participants: Ike, Varuna, Andrew, James
Notes: N/A
Results: Rescheduled our overarching goals based on our progress thus far.
Girls in Engineering Lab Tours
Participants: Liana, Aren
Notes: N/A
Results: Gave multiple lab tours to Girls in Engineering and showed them what a bioengineering lab was like.
Participants: Melody, Martina, Shayan, Marcella, Varuna, Rita
Notes: N/A
Results: Heifer gave us a lot of information Newcastle Disease and vaccines in resource constrained areas. We asked to partner with them and they said yes.
Meeting with Dean Wolfe
Participants: Marcella, Varuna
Notes: N/A
Results: Presented the project to the dean of Jack Baskin Engineering and received funding through the engineering department.
Outline Meeting
Participants: Ike, Varuna, Andrew, James
Notes: N/A
Results: Rescheduled our overarching goals based on our progress thus far.
Girls in Engineering Lab Tours
Participants: Liana, Aren
Notes: N/A
Results: Gave multiple lab tours to Girls in Engineering and showed them what a bioengineering lab was like.
Participants: Ike, Varuna, Andrew, James
Notes: N/A
Results: Rescheduled our overarching goals based on our progress thus far.
Girls in Engineering Lab Tours
Participants: Liana, Aren
Notes: N/A
Results: Gave multiple lab tours to Girls in Engineering and showed them what a bioengineering lab was like.
Wet Lab
Training in the Bio Safety Cabinet (BSC) began this week with practice of cleaning the BSC and proper aseptic technique using water. We also received HEK-293 cells from the Dubois Lab later this week to practice with. We received our primers for SDM and proceeded with mutating our plasmids. We ran the PCR products on a gel to verify which ones had succeeded.
Important Tasks
Miniprep of CAHS2 (D3)
Participants: Varuna, Martina
Protocols: Miniprep
Notes: We ran the liquid cultures using the Zymo Miniprep kit. This would allow us to verify the production of our plasmid in our transformed colonies.
Results: Nanodrop results indicated that the Miniprep was a success.
Site Directed Mutagenesis of CAHS 2 (D3), CAHS 1 (D4), & SAHS 10 (G3)
Participants: Andrew, Liana, Melody, Marcella, Martina, Varuna, Nikki
Protocols: Site Directed Mutagenesis Gel Electrophoresis
Notes: We plated 9 separate colonies onto index plates.
Results: We were able to grow these isolated colonies as separate indices to test for successful transformations.
Modeling of BL21 growth to find optimal point for Protein Production
Participants: Jonah, Iris
Protocols: N/A
Notes: 27 data points over 8 hours
Results: Results were graphed and used to develop our protein production protocols.
BSC Training
Participants: Jonah, Iris, James, Rita, Aren, Ike, Rita
Protocols: N/A
Notes: N/A
Results: Practiced proper aseptic technique with water and cleaning the BSC.
Dry Lab
Collaborations with other iGEM teams began this week with creating a storybook for the Vienna team and contacting the Stanford-Princeton-Brown team and the UC Davis team. We also began working on modeling and reevaluated our finances and medal requirements.
Significant Events:
Stanford-Princeton-Brown and Davis iGEM Meetup Coordination
Participants: Liana
Notes: Stanford-Princeton-Brown is one combined team working at Stanford.
Results: Got into contact with both teams about a possible meetup and collaboration.
Fundraising Meeting
Participants: Shayan, Marcella, Nikki, Liana
Notes: N/A
Results: We looked at our budget and how much money and reassessed goals and researched more contacts.
Vienna iGEM Collaboration: Children’s Book
Participants: Nikki
Notes: N/A
Results: Created a children’s story book called Violet the Super Vaccine to explain our project to children.
Meeting with Richelle Noroyan, UCSC Director of Community Outreach
Participants: Liana, Marcella, Melody, Nikki, Aren
Notes: N/A
Results: Introduced ourselves to the director of community outreach and confirmed meetings would be set up with various Rotary clubs in the area.
Met with Dr. Rebecca Dubois to Discuss Modeling our Proteins
Participants: Marcus, Shayan
Notes: N/A
Results: Dr. Dubois gave us a few different avenues to go down for modeling our proteins and verified our current model based on concentrations of IDP’s.
Participants: Shayan, Marcella, Nikki, Liana
Notes: N/A
Results: We looked at our budget and how much money and reassessed goals and researched more contacts.
Vienna iGEM Collaboration: Children’s Book
Participants: Nikki
Notes: N/A
Results: Created a children’s story book called Violet the Super Vaccine to explain our project to children.
Meeting with Richelle Noroyan, UCSC Director of Community Outreach
Participants: Liana, Marcella, Melody, Nikki, Aren
Notes: N/A
Results: Introduced ourselves to the director of community outreach and confirmed meetings would be set up with various Rotary clubs in the area.
Met with Dr. Rebecca Dubois to Discuss Modeling our Proteins
Participants: Marcus, Shayan
Notes: N/A
Results: Dr. Dubois gave us a few different avenues to go down for modeling our proteins and verified our current model based on concentrations of IDP’s.
Participants: Liana, Marcella, Melody, Nikki, Aren
Notes: N/A
Results: Introduced ourselves to the director of community outreach and confirmed meetings would be set up with various Rotary clubs in the area.
Met with Dr. Rebecca Dubois to Discuss Modeling our Proteins
Participants: Marcus, Shayan
Notes: N/A
Results: Dr. Dubois gave us a few different avenues to go down for modeling our proteins and verified our current model based on concentrations of IDP’s.
Wet Lab
This week's work consisted of finishing up the site directed mutagenesis on the flag tagged plasmids as well as testing our new electrocompetent cells. After finishing mutating all of our plasmids we transformed them back into DH5a cells to increase the amount of plasmid we had. We also began harvesting the HEK-293 cells in the BSL2 room for practice with cell counting and aseptic technique.
Site Directed Mutagenesis of CAHS 2 (D3), CAHS 1 (D4), SAHS 10 (G3), rvLEAM
Participants: James, Martina, Varuna, Ike, Shayan
Protocols: Site Directed Mutagenesis Gel Electrophoresis
Notes: N/A
Results: Gel showed ⅕ success. Only CAHS2 successful.
Site Directed Mutagenesis of CAHS 2 (D3), CAHS 1 (D4), rvLEAM (H4), LEA1
Participants: James, Andrew,Ike, Shayan
Protocols: Site Directed Mutagenesis Gel Electrophoresis
Notes: SDM was conducted using different Q5 master mixes to determine the reliability of each. Purpose - to add N terminal His tag
Results: 2 samples using the Q5 master mix were not successful, but a successful mutagenesis was obtained for every sample we ran.
Site Directed Mutagenesis of CAHS 7 (A2)
Participants: James, Andrew
Protocols: Site Directed Mutagenesis Gel Electrophoresis
Notes: Adding stop codon before 3’ His tag. Only needed to be done to this plasmid.
Results: Success
Transforming SAHS 10 (G3), CAHS 1 (D4), rvLEAM (H4), LEA1 (A4), CAHS 7 (A2) into DH5a cells.
Participants: James, Liana, Melody, Andrew
Protocols: Transformation
Notes: The mutagenized plasmids were transformed into DH5a E. coli and plated.
Results: LEA1, CAHS7 transformations failed. All others succeeded
Transforming LEA1 (A4), CAHS 7 (A2) into DH5a cells.
Participants: James, Melody
Protocols: Transformation
Notes: The mutagenized plasmids were transformed into DH5a E. coli and plated.
Results: LEA1 succeeded. CAHS7 failed.
Index Plating and Liquid Broth Inoculation of SDM transformed plasmids.
Participants: James, Melody
Protocols: Index Plating Liquid Inoculations
Notes: N/A
Results: We were able to grow these isolated colonies as separate indices to test for successful transformations.
Dry Lab
We held a small boba fundraiser outside of multiple buildings on campus to try and raise funds while meeting with students and faculty to discuss our project. We had the opportunity to meet the new chancellor through this and were able to set up a lab tour for them to see how iGEM works at UCSC. We also continued planning collaborations and talks with Heifer. We also created a second poster based on the first, containing our new results, for a presentation next week at the BD2K symposium.
Significant Events:
Boba Fundraiser
Participants: Everyone
Notes: Scheduled a lab tour with the chancelor and assistance chancelor for October.
Results: Raised roughly 350 dollars for the project and discussed our project with many students and faculty.
Contacting Doctors Without Borders
Participants: Melody
Notes: Doctors Without Borders is an international humanitarian organization known for its projects in countries affected by endemic diseases.
Results: Received articles and an issue brief on the importance of breaking the cold chain from doctors who had worked in resource constrained areas.
Participants: Melody
Notes: Doctors Without Borders is an international humanitarian organization known for its projects in countries affected by endemic diseases.
Results: Received articles and an issue brief on the importance of breaking the cold chain from doctors who had worked in resource constrained areas.
Wet Lab
The plasmids that had been mutated were miniprepped and sent out for sequencing. After analyzing the sequence data we stored all the plasmids that were successfully mutated away from the failures. CAHS7 was dropped at the point due to poor transformation efficiency. This could have been a result of having to mutate the plasmid twice. Cell maintenance and harvesting continued and a protein production protocol was developed to verify the heat solubility of the proteins.
Transformation of CAHS7
Participants: Andrew, James
Protocols: Transformation
Notes: N/A
Results: The transformation failed. Due to time constraints we opted to drop CAHS7 for the rest of our project and focus on the proteins that had been successful up to this point.
Miniprep of SAHS 10 (G3), CAHS 1 (D4), CAHS 2(D3), LEA1 (A4)
Participants: Andrew, Liana, James
Protocols: Miniprep
Notes: We ran the liquid cultures using the Zymo Miniprep kit. This would allow us to verify the production of our plasmid in our transformed colonies.
Results: Nanodrop results indicated that the Minipreps were successful. They were sent out for sequencing the next day.
Sent out miniprep of SAHS 10 (G3), CAHS 1 (D4), CAHS 2(D3), LEA1 (A4) for sequencing
Participants: Andrew, James
Protocols: N/A
Notes: We sent out plasmids to Sequetech for sequencing. Samples were run on a gel first.
Results: Sequencing data was reviewed and successful mutations were observed for every sample we sent out.
Index Plating and Liquid Broth Inoculation of rvLEAM (H4)
Participants: James, Melody
Protocols: Index Plating Liquid Inoculations
Notes: N/A
Results: We were able to grow these isolated colonies as separate indices to test for successful transformations.
Miniprep and Sequencing of rvLEAM(H4)
Participants: James, Melody
Protocols: Miniprep
Notes: N/A
Results: Nanodrop results indicated that the Minipreps were successful. We verified the successful insert of the 6X His-tag.
Protein Production of CAHSD-Pilot
Participants: Jonah, Iris
Protocols: Day 1 Day 2 Purification
Notes: N/A
Results: Successful.
Dry Lab
This week we gave multiple presentations on our project to the UCSC community. We also received a new contact at Genentech that would benefit us later. We were also able to share the children’s book that had been developed with more people through the Monterey SPCA.
Significant Events:
BD2K Symposium Poster Presentation
Participants: Ike, Nikki, Liana, Andrew, Iris
Notes: N/A
Results: Had an opportunity to share the scientific research, the human practices, and the current results of our project with the UCSC community.
Biolink Depot
Participants: James, Shayan, Ike
Notes: N/A
Results: Received a significant grant from Porter College.
Meeting with Porter Provost
Participants: Martina, Shayan
Notes: Road Trip!
Results: Acquired many lab supplies for our experiments.
Bioethics 80G Presentation
Participants: Shayan, Marcella, Rita, Martina, Andrew, Ike
Notes: N/A
Results: Presented our project and held a Q&A at a bioethics class at UCSC. We discussed the underlying ethical principles of the projects and the issues that might arise from it.
Monterey Society for the Prevention of Cruelty to Animals
Participants: Martina, Melody
Notes: N/A
Results: Shared the Children’s book Nikki made with them to be implemented in educating children about the importance of vaccinating animals in their 2020 summer camp.
Participants: James, Shayan, Ike
Notes: N/A
Results: Received a significant grant from Porter College.
Meeting with Porter Provost
Participants: Martina, Shayan
Notes: Road Trip!
Results: Acquired many lab supplies for our experiments.
Bioethics 80G Presentation
Participants: Shayan, Marcella, Rita, Martina, Andrew, Ike
Notes: N/A
Results: Presented our project and held a Q&A at a bioethics class at UCSC. We discussed the underlying ethical principles of the projects and the issues that might arise from it.
Monterey Society for the Prevention of Cruelty to Animals
Participants: Martina, Melody
Notes: N/A
Results: Shared the Children’s book Nikki made with them to be implemented in educating children about the importance of vaccinating animals in their 2020 summer camp.
Participants: Shayan, Marcella, Rita, Martina, Andrew, Ike
Notes: N/A
Results: Presented our project and held a Q&A at a bioethics class at UCSC. We discussed the underlying ethical principles of the projects and the issues that might arise from it.
Monterey Society for the Prevention of Cruelty to Animals
Participants: Martina, Melody
Notes: N/A
Results: Shared the Children’s book Nikki made with them to be implemented in educating children about the importance of vaccinating animals in their 2020 summer camp.
Wet Lab
This week marked the start of large scale production as well as the production of other plasmids such as SAHS10 that were transformed into Bl21 cells earlier in the week. BSL2 training continued as usual with cell harvesting.
CAHS D Large Scale Protein Production
Participants: Melody, Preet, Martina, Jonah
Protocols: Protein Production Large Scale
Notes: No tags present on protein.
Results: Success.
Miniprep of SDM DH5a colonies and transformation in BL21 cells for SAHS 10 (G3), CAHS 1 (D4), CAHS 2(D3), LEA1 (A4)
Participants: Liana, James, Andrew, Martina
Protocols: Miniprep , Transformation
Notes: Additional minipreps were necessary as we had used up most of our stocks on sequencing and gel electrophoresis. The remainder of these stocks were stored away from the others.
Results: Successful minipreps and transformations.
Protein Production & Purification of SAHS 10 (G3) - Pilot
Participants: Melody, Jonah, Martina
Protocols: Day 1 Day 2 Purification
Notes: N/A
Results: No Protein Produced - Failure
Dry Lab
This week was finally the week of the Stanford-Brown-Princeton meetup and though Davis was not able to attend it was a great experience to share the successes and difficulties of our project with those who had similar experiences. We also started presenting our project to local Rotary Clubs in the area. Finally, we got in contact with the 4H clubs in the area who gave us the contact information of Terry Reader who would get us a table at the Santa Cruz County Fair next month.
Significant Events:
Sunrise Rotary Presentation
Participants: Shayan, Marcella, Liana
Notes: N/A
Results: Our PI Dr. David Bernick gave a presentation on our project to the sunrise rotary club and we answered questions that they had.
Meeting with Dean Koch of PBsci
Participants: Nikki, Marcella, Shayan, Aren
Notes: N/A
Results: Recieved a significant grant for our project.
Stanford-Brown-Princeton Meetup
Participants: Liana, Andrew, Martina, Melody, Shayan, Marcella
Notes: Davis was not able to attend
Results: Had the opportunity to discuss each others projects.
Participants: Nikki, Marcella, Shayan, Aren
Notes: N/A
Results: Recieved a significant grant for our project.
Stanford-Brown-Princeton Meetup
Participants: Liana, Andrew, Martina, Melody, Shayan, Marcella
Notes: Davis was not able to attend
Results: Had the opportunity to discuss each others projects.
Wet Lab
This week consisted entirely of protein production which all resulted in failures. This led to the troubleshooting meeting the following week.
Protein Production & Purification of rvLEAM (H4) - Pilot
Participants: James, Marcella
Protocols: Day 1 Day 2 , Purification
Notes: Improper protein production methods. → Troubleshooting
Results: No Protein Produced
Re-do Protein Production & Purification of SAHS 10 (G3) - Pilot
Participants: Melody, Martina
Protocols: Day 1 Day 2 Purification
Notes: Improper protein production methods. → Troubleshooting
Results: Results inconclusive again
Dry Lab
The second draft of the technical report was finished this week with new results and human practices that had occured the last five weeks. We also set up an interview with Rose Miyatsu, a communications assistant at Jack Baskin School of Engineering, for a story about iGEM. Finally, we also had a protein production troubleshooting meeting and isolated some possible suspects that were causing our experiments to fail.
Significant Events:
EOP Meeting
Participants: Nikki, Marcella
Notes: N/A
Results: Received funding and established iGEM-EOP relations.
Protein Production Troubleshooting Meeting
Participants: Everyone
Notes: N/A
Results: The entire team got together and attempted to find out what was causing our protein production to fail. We had a few suspects, including cell identity, and bad IPTG.
Set up iGEM Feature Story for the UCSC Campus Newsletter
Participants: Shayan
Notes: N/A
Results: Rose Miyatsu contacted us to set up a group interview for us to talk about our research. She contacted us on behalf of the Baskin School of Engineering
Participants: Everyone
Notes: N/A
Results: The entire team got together and attempted to find out what was causing our protein production to fail. We had a few suspects, including cell identity, and bad IPTG.
Set up iGEM Feature Story for the UCSC Campus Newsletter
Participants: Shayan
Notes: N/A
Results: Rose Miyatsu contacted us to set up a group interview for us to talk about our research. She contacted us on behalf of the Baskin School of Engineering
Wet Lab
This week consisted of troubleshooting various aspects of our protein production including the cell type, buffers, and heat shock temperature. This week we also attempted to develop a primary cell line from eggs donated to us by a local farm, but all of the eggs were not fertilized. A small subset of the team was taught a Western blot protocol by Dr. Dubois.
Troubleshoot Protein Production & Purification of SAHS 10 (G3) - Pilot
Participants: Melody, Jonah, Martina
Protocols: Day 1 Day 2 Purification
Notes: Changed Heat Shock temperature, re-made all buffers.
Results: Inconclusive again. Further troubleshooting is required.
Confirmed that all cells used in protein production are BL21 cells.
Participants: Andrew, James
Protocols: N/A
Notes: The cells were autolysed using arabinose.
Results: Cells were lysed as we would expect if they were bl21 cells.
Creating Primary Cell Lines from Embryonated Chicken Eggs
Participants: Jonah, Iris, Preet, Aren
Protocols: N/A
Notes: Eggs were donated from local farm.
Results: Failed. No embryos, eggs were not fertilized.
Western Blot (Test) double check date
Participants: Jonah, Iris, Preet, Martina
Protocols: N/A
Notes: To quantify proteins.
Results: Failed. No proteins showed up on the membrane.
Dry Lab
Along with another rotary presentation, a small group of the team went to a local biotech conference and connected with members of the biotech community. Finally, relations with Terry Reeder continued and led to the setup of a booth at the Santa Cruz County Fair.
Significant Events:
Santa Cruz Tech Meetup
Participants: Marcella, Shayan, Rita
Notes: Local Biotech Conference
Results: Shayan did a 1 minute talk during the company presentations portion of the conference. Establish relations with the local biotech community. Talked with people before the presentations at a table set up for us.
Santa Cruz Rotary Presentation
Participants: James, Aren, Marcella, Rita
Notes: Rotary of Santa Cruz
Results: Presented our project to rotarians and answered questions.
Modeling research
Participants: Rita, Shayan
Notes: Using the radius of gyration might be the best way to properly get concentration
Results: More research must be done
Participants: James, Aren, Marcella, Rita
Notes: Rotary of Santa Cruz
Results: Presented our project to rotarians and answered questions.
Modeling research
Participants: Rita, Shayan
Notes: Using the radius of gyration might be the best way to properly get concentration
Results: More research must be done
Wet Lab
At this point we finally identified the problem with our protein production methods. It turns out the 6XHis-tags were causing a hairpin to form leading to no expression. We quickly fixed the primer issue and ordered new primers. To verify this, we tested the FLAG tagged proteins and after getting successful expression we knew the hairpin was the culprit.
Problem with SDM primers were identified. Protein production with our his tags were failing due to hairpin formation, preventing transcription. Only our untagged plasmid (CAHS D) was successful up to this point
Participants: Shayan
Protocols: N/A
Notes: Protein production with our His tags were failing due to hairpin formation, preventing transcription. Only our untagged plasmid (CAHS D) was successful up to this point.
Results: New Primers designed and ordered
Protein Production of FLAG tagged CAHS 1 Plasmid
Participants: Jonah, Iris, Aren
Protocols: Day 1 Day 2 Purification
Notes: To test if primers did indeed form a hairpin and prevent transcription of the protein.
Results: Successfully produced CAHS1.
Dry Lab
This week we went to the Santa Cruz County Fair and shared our project with those that worked directly with animals. We also had an interview with Rose Miyatsu about our project. The results of this interview were later published on the UCSC Newsletter Website.
Significant Events:
County Fair
Participants: Everyone
Notes: Everyone took at least one shift at the County Fair from Wednesday to Sunday.
Results: We were able to share our project with people at the farm and received duck eggs from a generous donor.
Interview with Rose Miatsu
Participants: Everyone
Notes: Rose came in and asked us about our project, our experiences as students, and what we would like to tell the UCSC community.
Results: The results of the interview were published in an article on the UCSC Newsletter Website.
Participants: Everyone
Notes: Rose came in and asked us about our project, our experiences as students, and what we would like to tell the UCSC community.
Results: The results of the interview were published in an article on the UCSC Newsletter Website.
Wet Lab
This week was devoted to re-doing site directed mutagenesis to all our plasmids in order to rectify the hairpin problem our previous plasmids had. Large scale production was performed on all of the FLAG tagged proteins and succeeded. However, they were not used due to their impurity.
SDM Using New Primers and Gel Electrophoresis for CAHS1(D4),CAHS2(D3), LEA1(A4), rvLEAM(H4), SAHS10(G3)
Participants: James, Shayan
Protocols: Site Directed Mutagenesis Gel Electrophoresis
Notes: N/A
Results: Added a His Tag to successful plasmids, of which all had at least one.
Transformation of CAHS1(D4),CAHS2(D3), LEA1(A4), rvLEAM(H4), SAHS10(G3) into DH5a cells
Participants: James, Melody, Martina
Protocols:
Notes: N/A
Results: Successful transformation of all plasmids into E. coli cells.
SDM of CAHSD
Participants: Shayan, James, Melody
Protocols: Site Directed Mutagenesis Gel Electrophoresis
Notes: CAHSD was done at a different time because it had different primers.
Results: Successful mutagenesis.
Large Protein Production and Purification of FLAG tagged CAHS1(D4),CAHS2(D3), LEA1(A4), rvLEAM(H4), SAHS10(G3)
Participants:Iris, Jonah, Aren, Preet
Protocols: Protein Production Large Scale
Notes: Purification steps were changed to heat solubilize proteins in the autoclave instead of using a 95C water bath
Results: Success. Surprisingly enough, all of the proteins except for rvLEAM survived the autoclave.
Plaque Assay of LaSota Strain of NDV
Participants: Iris, Jonah, Aren, Preet
Protocols:
Notes: N/A
Results: Failed.
Dry Lab
Liana began visiting schools to educate them on our project and get them excited for the possibilities of STEM. Modeling research continued, but the suggestions given to us would require a significant amount of time and money. A larger subset of the team began working to finish the website before the deadline.
Significant Events:
Harbor Highschool Visit and DeLaveaga Elementary School Visit
Participants: Liana
Notes: N/A
Results: Presented our project and educated them on the importance of breaking the cold chain and animal vaccination.
Genentech Email
Participants: Ike
Notes: N/A
Results: Got into contact with Don Kirkpatrick Associate Director at Genentech. Scheduled a meeting for later that month.
Meeting with Dr. Mcshan regarding modeling
Participants: Rita, Shayan, Marcella
Notes: Hydrogel analysis and using SAXS or a computer simulation might be the best option for an accurate model for concentration. Not much biophysical characterization has been made on IDP’s. Very good resource for information.
Results: The planned model would be too time consuming to do properly in this time frame so an alternative would need to be found.
Participants: Ike
Notes: N/A
Results: Got into contact with Don Kirkpatrick Associate Director at Genentech. Scheduled a meeting for later that month.
Meeting with Dr. Mcshan regarding modeling
Participants: Rita, Shayan, Marcella
Notes: Hydrogel analysis and using SAXS or a computer simulation might be the best option for an accurate model for concentration. Not much biophysical characterization has been made on IDP’s. Very good resource for information.
Results: The planned model would be too time consuming to do properly in this time frame so an alternative would need to be found.
Wet Lab
Plasmids were miniprepped and sent out for sequencing. All the SDM plasmids were successful and we began transforming them into BL21 cells so we could begin protein production. We also received 13 embryonated chicken eggs which we incubated and turned three times a day. These would be inoculated with virus once they reached day 10.
Index Plates and Liquid Cultures of CAHS1(D4),CAHS2(D3), LEA1(A4), rvLEAM(H4), SAHS10(G3)
Participants N/A
Protocols: Liquid Inoculation
Notes: N/A
Results: Grew up liquid cultures of all of our mutated plasmids to be miniprepped the next day.
Minipreps and Sequencing of SDM Plasmids CAHS1(D4),CAHS2(D3), LEA1(A4), rvLEAM(H4), SAHS10(G3)
Participants: James, Melody
Protocols: Miniprep
Notes: N/A
Results: Successful. Plasmids were sent out for sequencing the next day.
Transformation of SDM’s into BL21 Cells
Participants: Melody, Martina, Varuna, Marcella
Protocols: Transformation
Notes: Transformed all plasmids into BL21 cells before we had sequencing data to save time.
Results: Transformations were successful.
Analysing Sequencing Data of Plasmids
Participants: James
Protocols:
Notes: Threw out all of the plates that didn’t have good reads.
Results: LEA1(A4), rvLEAM(H4), SAHS10(G3), and CAHSD were successful, but CAHS1(D4) was missing a singular base in the His-tag and CAHS2(D3) had been mixed or contaminated with SAHS10(G3).
Arrival of 13 Embryonated Chicken Eggs
Participants: N/A
Protocols: N/A
Notes: One egg arrived cracked
Results: Began incubating the eggs and turning them three times a day.
Protein Production of His Tagged SAHS10(G3), LEA1(A4)-Pilot
Participants: James, Melody, Martina
Protocols: Day 1 Day 2 Purification
Notes: Purification steps were changed to heat solubilize proteins in the autoclave instead of using a 95C water bath
Results: Gels were too faded to tell if results were good or not. Started protein production again.
Protein Production of His tagged CAHSD-Pilot
Participants: Varuna, Andrew, Ike
Protocols: Day 1 Day 2 Purification
Notes: N/A
Results: Successful.
SDM Second Round of CAHS1
Participants: Shayan, James
Protocols: Site Directed Mutagenesis Gel Electrophoresis
Notes: N/A
Results: Successful.
Transformation, Index Plating, and Liquid Cultures of CAHS1(D4)
Participants: James, Melody, Martina
Protocols:
Notes: N/A
Results: Successful. Stored to be miniprepped next week.
Analysis of SDM CAHS2 Plasmid (D3), Transformation Plates, and Index Plates by Sequencing
Participants: James
Protocols: Site Directed Mutagenesis Gel Electrophoresis
Notes: Miniprepped the transformation and index plates and the original plasmid to verify where contaminated CAHS2(D3) and SAHS10(G3).
Results: Original plasmid was fine, transformation plates were not so we mutated and transformed the wrong plasmid.
Notes: Threw out all of the plates that didn’t have good reads.
Results: LEA1(A4), rvLEAM(H4), SAHS10(G3), and CAHSD were successful, but CAHS1(D4) was missing a singular base in the His-tag and CAHS2(D3) had been mixed or contaminated with SAHS10(G3).
Arrival of 13 Embryonated Chicken Eggs
Participants: N/A
Protocols: N/A
Notes: One egg arrived cracked
Results: Began incubating the eggs and turning them three times a day.
Protein Production of His Tagged SAHS10(G3), LEA1(A4)-Pilot
Participants: James, Melody, Martina
Protocols: Day 1 Day 2 Purification
Notes: Purification steps were changed to heat solubilize proteins in the autoclave instead of using a 95C water bath
Results: Gels were too faded to tell if results were good or not. Started protein production again.
Protein Production of His tagged CAHSD-Pilot
Participants: Varuna, Andrew, Ike
Protocols: Day 1 Day 2 Purification
Notes: N/A
Results: Successful.
SDM Second Round of CAHS1
Participants: Shayan, James
Protocols: Site Directed Mutagenesis Gel Electrophoresis
Notes: N/A
Results: Successful.
Transformation, Index Plating, and Liquid Cultures of CAHS1(D4)
Participants: James, Melody, Martina
Protocols:
Notes: N/A
Results: Successful. Stored to be miniprepped next week.
Analysis of SDM CAHS2 Plasmid (D3), Transformation Plates, and Index Plates by Sequencing
Participants: James
Protocols: Site Directed Mutagenesis Gel Electrophoresis
Notes: Miniprepped the transformation and index plates and the original plasmid to verify where contaminated CAHS2(D3) and SAHS10(G3).
Results: Original plasmid was fine, transformation plates were not so we mutated and transformed the wrong plasmid.
Dry Lab
Liana continued to visit schools and present our project. Later in the week iGEM set up a booth at the UCSC Cornucopia, a club festival, where we showcased iGEM and recruited for next years iGEM team.
Significant Events:
Branciforte Middle School Visit
Participants: Liana
Notes: N/A
Results: Presented our project and educated them on the importance of breaking the cold chain and animal vaccination.
UCSC 2019 Cornucopia
Participants: Liana, Shayan, Marcella, James, Nikki, Rita, Melody, Andrew, Martina
Notes: A festival where all the clubs on campus recruit and show students what they are about
Results: Shared our project with various students and spread the word about next year’s iGEM.
Facetime Meeting with Don Kirkpatrick
Participants: Ike
Notes: N/A
Results: Ike shared our project and it was then shared with Sarah Hymowitz of Genentech.
Participants: Liana, Shayan, Marcella, James, Nikki, Rita, Melody, Andrew, Martina
Notes: A festival where all the clubs on campus recruit and show students what they are about
Results: Shared our project with various students and spread the word about next year’s iGEM.
Facetime Meeting with Don Kirkpatrick
Participants: Ike
Notes: N/A
Results: Ike shared our project and it was then shared with Sarah Hymowitz of Genentech.
Wet Lab
Plaque Assays continued and were unsuccessful. Troubleshooting began the next week. Site directed mutagenesis finished with rerunning CAHS2, and CAHS1 was sent out for sequencing. We finally got success with protein production with pilots of CAHSD, LEA1, and SAHS10 and even finished purifying a large scale version of CAHSD.
Plaque Assay Trials (BSL2)
Participants: Iris, Jonah, Aren, Preet
Protocols: Plaque Assay
Notes: Preparing and testing the protocol for Plaque Assays.
Results: Unsuccessful.
Miniprep, Sequencing, and Sequence Analysis of CAHS1(D4)
Participants: James, Melody
Protocols:
Notes: N/A
Results: Successfully verified the SDM of CAHS1.
Transformation of CAHS1(D4) into BL21 cells
Participants: James,
Protocols:
Notes: N/A
Results: Successful.
SDM of CAHS2(D3) second round
Participants: James
Protocols: Site Directed Mutagenesis Gel Electrophoresis
Notes: N/A
Results: Successful.
Transformation of CAHS2(D3) into DH5a cells and Index Plating
Participants: James, Melody
Protocols:
Notes: N/A
Results: Successful.
Protein Production of His Tagged SAHS10(G3), LEA1(A4)-Pilot 2nd Attempt
Participants: James, Melody, Martina
Protocols: Day 1 Day 2 Purification
Notes: N/A
Results: Success
Protein Production His TaggedCAHSD-Large Scale
Participants: Varuna, Andrew
Protocols: Protein Production Large Scale
Notes: CAHSD had a huge band!
Results: Successful.
Miniprep of His Tagged CAHS2(D3)
Participants: James, Melody
Protocols:
Notes: N/A
Results: Failed. DNA yields extremely low. Attempted again same issue. Began troubleshooting.
Dry Lab
Besides the Rotary Club presentation all of our work in the dry lab was spent working on the Wiki.
Significant Events:
Wiki Work Education, Outreach, Public Engagement, Human Practices and The Notebook Pages:
Participants: Liana, Marcus, James, Shayan, Andrew, Nikki, Marcella
Notes: N/A
Results: Significant effort was spent on developing the above pages for the website before the October deadline.
Scotts Valley Rotary Presentation
Participants: Marcella, Rita, Shayan
Notes: N/A
Results: Rita presented on our project.
Participants: Marcella, Rita, Shayan
Notes: N/A
Results: Rita presented on our project.
Wet Lab
Troubleshooting on Plaque Assays continued. Once the eggs had reached day 10 they were injected with dilutions of the vaccine in order to propagate the virus. New Zyppy wash buffer was created to try and fix the Minipreps, but to no avail.
Troubleshooting Plaque Assays
Participants:Jonah, Iris, Aren, Preet
Protocols: Attempted different levels of cell confluency and trypsin concentrations.
Notes: N/A
Results: Plaque Assays still unsuccessful.
Protein Production of His Tagged CAHS1(D4)-Pilot
Participants: James
Protocols: Protein Production
Notes: N/A
Results: Successful.
Large Scale Production and Purification of CAHS1(D4), LEA1(A4), SAHS10(G3)
Participants: James, Melody, Martina
Protocols: Protein Production Large Scale
Notes: Insoluble fractions were loaded with too much protein and caused the gel to streak.
Results: Successful.
SDS PAGE Gel Comparing CAHS1(D4), CAHSD, LEA1(A4), and SAHS10(G3)
Participants: James, Melody,
Protocols:
Notes: Re-ran the insoluble fractions due to smudges on previous gel and comparing relative expression levels of the various proteins.
Results: A very nice gel comparing the proteins.
Dry Lab
Work on the Wiki continued and we were able to give a lab tour to the Chancellor and Assistant Chancellor.
Significant Events:
Response from Terry on Questionnaire about Chicken Vaccination and Newcastle
Participants: Martina
Notes: N/A
Results: Terry responded to our questions within an hour. It gave us information on how people who own chickens experience and deal with Newcastle Disease.
Lab Tour for the Chancellor and Assistant Chancellor
Participants: onah, Aren, Iris, James, Melody, Liana, Martina, Shayan, Marcella
Notes: N/A
Results: Showed our chancellor the importance of our project and how much it teaches undergraduates about research.
Participants: onah, Aren, Iris, James, Melody, Liana, Martina, Shayan, Marcella
Notes: N/A
Results: Showed our chancellor the importance of our project and how much it teaches undergraduates about research.
Wet Lab
Purification of the successful large scales began this week with CAHS1. A new miniprep kit was given to us by the Dubois Lab, but again resulted in failure. This meant that our technique was likely the culprit and so we held a troubleshooting meeting with David to address some of the potential mistakes. Protein Production was attempted on rvLEAM which had failed when FLAG tagged so we expected it to fail again. Allantoic fluid, hopefully containing the virus was harvested from the inoculated eggs and frozen. Finally, we received pig blood and attempted to harvest red blood cells from it for hemagglutination assays but failed.
Protein Production & Purification of His Tagged rvLEAM (H4) - Pilot
Participants: Marcella, James, Martina
Protocols: Day 1 Day 2 Purification
Notes: Improper protein production methods. → Troubleshooting
Results: No Protein Produced
Harvesting of Red Blood Cells from Pig’s Blood
Participants: Martina, Jonah, Aren, Iris, Preet
Protocols:
Notes: Something was added to the blood to prevent coagulation
Results: Failed, cell counts were extremely low. This could be because of whatever was added to the blood
Purification of His Tagged CAHS1(D4) on the His column
Participants: James, Melody, Martina
Protocols: Histidine Column Purification
Notes: Because the supernatant was not filtered before use the column was clogged and took over 20 hours. In addition, it appears we overloaded the column as there was still a significant amount of protein that did not bind.
Results: Purified CAHS1 away from DNA and the remaining proteins. Imidazole concentrations need to be tweaked for more optimal elution.
Dry Lab
Significant Events:
Wiki Work
Participants: Everyone
Notes: N/A
Results: Finished putting up wiki.
Donation from the Hymowitz Foundation
Participants: Ike
Notes: N/A
Results: Received a substantial donation from the Hymowitz Foundation. Thank you so much.
Participants: Ike
Notes: N/A
Results: Received a substantial donation from the Hymowitz Foundation. Thank you so much.
Wet Lab
The last week before the wiki freeze was a rush to finish things. Miniprep kits finally started working again and so CAHS2 was sent out to be sequenced, transformed into BL21 and protein production was attempted. Unfortunately, it failed and no protein was produced. Plaque assays may have been fixed by adding more media, but the results are still to come. A hemagglutination assay was attempted.
Miniprep and Sequencing of His Tagged CAHS2(D3)
Participants: Martina, James, Melody
Protocols:
Notes: N/A
Results: Miniprep successful. Used two rounds of Zyppy wash buffer to remove more salt. Sequencing data also showed the plasmid was mutated successfully.
Transformation into BL21, Index Plates, and Liquid Cultures of His Tagged CAHS2(D3)
Participants: Martina, James,
Protocols:
Notes: N/A
Results: Successfully transformed and prepared for protein production.
Protein Production & Purification of His Tagged CAHS2(D3) - Pilot
Participants: Marcella, Ike, James
Protocols: Day 1 Day 2 Purification
Notes: N/A
Results: No Protein Produced.
Plaque Assays
Participants: Aren, Jonah, Iris
Protocols: Plaque Assays
Notes: Tried different amounts of media
Results: Partial success. Cells were now sticking to the plate and being dyed successfully.
Harvesting of Red Blood Cells from Turkey Blood
Participants: Martina, Jonah, Aren, Iris, Preet
Protocols:
Notes: N/A
Results: Success. Cell counts were within the estimates. We could now continue with hemagglutination assays.
Hemagglutination Assay
Participants: Aren, Jonah, Iris
Protocols:
Notes: N/A
Results: TBD
Purification of His Tagged CAHSD, LEA1(A4), and SAHS10 on the His Column
Participants: James, Melody, Varuna, Martina
Protocols: Histidine Column Purification
Notes: Gel for CAHSD broke a bit.
Results: Success for SAHS10 and CAHSD. LEA1 failed.
