iGEM 2019 — Team UCSC

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# Team:UCSC/Notebook

[![](https://static.igem.org/mediawiki/2019/c/c2/T--UCSC--vitrum-logo-offwhite.png)](https://2019.igem.org/Team%3AUCSC)

* Team▾
  + [Team](https://2019.igem.org/Team%3AUCSC/Team)
  + [Attributions](https://2019.igem.org/Team%3AUCSC/Attributions)
  + [Collaborations](https://2019.igem.org/Team%3AUCSC/Collaborations)
* Project ▾
  + [Description](https://2019.igem.org/Team%3AUCSC/Description)
  + [Design](https://2019.igem.org/Team%3AUCSC/Design)
  + [Experiments](https://2019.igem.org/Team%3AUCSC/Experiments)
  + [Notebook](https://2019.igem.org/Team%3AUCSC/Notebook)
  + [Results](https://2019.igem.org/Team%3AUCSC/Results)
* Parts ▾
  + [Parts](https://2019.igem.org/Team%3AUCSC/Parts)
* [Safety](https://2019.igem.org/Team%3AUCSC/Safety)
* Human Practices ▾
  + [Integrated](https://2019.igem.org/Team%3AUCSC/Human_Practices)
  + [Engagement & Education](https://2019.igem.org/Team%3AUCSC/Public_Engagement)
  + [Entrepreneurship](https://2019.igem.org/Team%3AUCSC/Entrepreneurship)

#### Notebook

June

+ June 24 - 30

# 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.

# 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.

July

+ July 1 - 7

# 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.

# 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](https://static.igem.org/mediawiki/2019/c/cb/T--UCSC--plasmid.pdf) [Transformation](https://static.igem.org/mediawiki/2019/3/3f/T--UCSC--transformation-notebook.pdf)

#### 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](https://static.igem.org/mediawiki/2019/8/89/T--UCSC--index-plating.pdf)

#### 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)](https://static.igem.org/mediawiki/2019/8/89/T--UCSC--index-plating.pdf)

#### 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.

+ July 8 - 14

# 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.

# 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](https://static.igem.org/mediawiki/2019/0/0c/T--UCSC--zyppy.pdf) , [Transformation](https://static.igem.org/mediawiki/2019/3/3f/T--UCSC--transformation-notebook.pdf)

#### 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](https://static.igem.org/mediawiki/2019/5/53/T--UCSC--gel-electro.pdf) 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.

![](https://static.igem.org/mediawiki/2019/7/70/T--UCSC--notebook1.png)
![](https://static.igem.org/mediawiki/2019/5/5a/T--UCSC--notebook2.jpg)

### 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](https://static.igem.org/mediawiki/2019/c/cb/T--UCSC--plasmid.pdf) , [Transformation](https://static.igem.org/mediawiki/2019/3/3f/T--UCSC--transformation-notebook.pdf)

#### 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](https://static.igem.org/mediawiki/2019/c/cb/T--UCSC--plasmid.pdf), [Transformation](https://static.igem.org/mediawiki/2019/3/3f/T--UCSC--transformation-notebook.pdf)

#### 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](https://static.igem.org/mediawiki/2019/3/3f/T--UCSC--transformation-notebook.pdf)

#### 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](https://static.igem.org/mediawiki/2019/c/cb/T--UCSC--plasmid.pdf) , [Transformation](https://static.igem.org/mediawiki/2019/3/3f/T--UCSC--transformation-notebook.pdf)

#### 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](https://static.igem.org/mediawiki/2019/8/89/T--UCSC--index-plating.pdf)

#### 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](https://static.igem.org/mediawiki/2019/c/cb/T--UCSC--plasmid.pdf) , [Transformation](https://static.igem.org/mediawiki/2019/3/3f/T--UCSC--transformation-notebook.pdf)

#### 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](https://static.igem.org/mediawiki/2019/8/89/T--UCSC--index-plating.pdf), 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.

![](https://static.igem.org/mediawiki/2019/c/c4/T--UCSC--third.jpg)

### Index Plating, Colony PCR, and Gel electrophoresis of CAHS D

#### Participants: Iris, Jonah

#### Protocols: [Index Plating & Colony PCR](https://static.igem.org/mediawiki/2019/8/89/T--UCSC--index-plating.pdf), 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.

![](https://static.igem.org/mediawiki/2019/5/56/T--UCSC--fourth.jpg)

### Sterilizing BSL2:

#### Participants: Iris, Jonah

#### Protocols: N/A

#### Notes: N/A

#### Results: Room and equipment were sterilized.

+ July 15 - 21

# 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.

# 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](https://static.igem.org/mediawiki/2019/c/cb/T--UCSC--plasmid.pdf) , [Transformation](https://static.igem.org/mediawiki/2019/3/3f/T--UCSC--transformation-notebook.pdf)

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](https://static.igem.org/mediawiki/2019/8/89/T--UCSC--index-plating.pdf) and [Gel Electrophoresis](https://static.igem.org/mediawiki/2019/5/53/T--UCSC--gel-electro.pdf)

#### Results: All colony PCR samples showed the expected insert fragment length.

![](https://static.igem.org/mediawiki/2019/8/8f/T--UCSC--fifth.jpg)

### Miniprep of CAHS1 (D4), SAHS 10 (G3) & rvLEAM (H4)

#### Participants: Andrew, Liana, Melody, Nikki, Marcella

#### Protocols: [Miniprep](https://static.igem.org/mediawiki/2019/0/0c/T--UCSC--zyppy.pdf)

#### 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](https://static.igem.org/mediawiki/2019/5/53/T--UCSC--gel-electro.pdf)

#### Results: Gel showed major streaking. It was concluded that Colony 6 would be used for minipreps.

![](https://static.igem.org/mediawiki/2019/d/d3/T--UCSC--image11.jpg)

### Gel electrophoresis of CAHS1 (D4), SAHS 10 (G3) & rvLEAM (H4)

#### Participants: Andrew, Melody

#### Protocols: Colony PCR [Gel Electrophoresis](https://static.igem.org/mediawiki/2019/5/53/T--UCSC--gel-electro.pdf)

#### Notes: N/A

#### Results: Lengths of the purified plasmids were confirmed and correct.

![](https://static.igem.org/mediawiki/2019/1/11/T--UCSC--image7.jpg)

+ July 22 - 28

# 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.

# 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](https://static.igem.org/mediawiki/2019/0/0c/T--UCSC--zyppy.pdf)

#### 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](https://static.igem.org/mediawiki/2019/5/59/T--UCSC--mutagenesis.pdf) [Gel Electrophoresis](https://static.igem.org/mediawiki/2019/5/53/T--UCSC--gel-electro.pdf)

#### 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.

![](https://static.igem.org/mediawiki/2019/7/7d/T--UCSC--image5.jpg)

### 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.

August

+ July 29 - August 4

# 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.

# 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](https://static.igem.org/mediawiki/2019/5/59/T--UCSC--mutagenesis.pdf) [Gel Electrophoresis](https://static.igem.org/mediawiki/2019/5/53/T--UCSC--gel-electro.pdf)

#### Notes: N/A

#### Results: Gel showed ⅕ success. Only CAHS2 successful.

![](https://static.igem.org/mediawiki/2019/9/9a/T--UCSC--NotebookScreenshot1.png)

### Site Directed Mutagenesis of CAHS 2 (D3), CAHS 1 (D4), rvLEAM (H4), LEA1

#### Participants: James, Andrew,Ike, Shayan

#### Protocols: [Site Directed Mutagenesis](https://static.igem.org/mediawiki/2019/5/59/T--UCSC--mutagenesis.pdf) [Gel Electrophoresis](https://static.igem.org/mediawiki/2019/5/53/T--UCSC--gel-electro.pdf)

#### 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.

![](https://static.igem.org/mediawiki/2019/9/9e/T--UCSC--image4.png)

### Site Directed Mutagenesis of CAHS 7 (A2)

#### Participants: James, Andrew

#### Protocols: [Site Directed Mutagenesis](https://static.igem.org/mediawiki/2019/5/59/T--UCSC--mutagenesis.pdf) [Gel Electrophoresis](https://static.igem.org/mediawiki/2019/5/53/T--UCSC--gel-electro.pdf)

#### Notes: Adding stop codon before 3’ His tag. Only needed to be done to this plasmid.

#### Results: Success

![](https://static.igem.org/mediawiki/2019/4/47/T--UCSC--image8.png)

### Transforming SAHS 10 (G3), CAHS 1 (D4), rvLEAM (H4), LEA1 (A4), CAHS 7 (A2) into DH5a cells.

#### Participants: James, Liana, Melody, Andrew

#### Protocols: [Transformation](https://static.igem.org/mediawiki/2019/3/3f/T--UCSC--transformation-notebook.pdf)

#### 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](https://static.igem.org/mediawiki/2019/3/3f/T--UCSC--transformation-notebook.pdf)

#### 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](https://static.igem.org/mediawiki/2019/8/89/T--UCSC--index-plating.pdf) Liquid Inoculations

#### Notes: N/A

#### Results: We were able to grow these isolated colonies as separate indices to test for successful transformations.

+ August 5 - 11

# 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.

# 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](https://static.igem.org/mediawiki/2019/3/3f/T--UCSC--transformation-notebook.pdf)

### 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.

![](https://static.igem.org/mediawiki/2019/6/62/T--UCSC--image3.png)

## Index Plating and Liquid Broth Inoculation of rvLEAM (H4)

### Participants: James, Melody

### Protocols: [Index Plating](https://static.igem.org/mediawiki/2019/8/89/T--UCSC--index-plating.pdf) 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](https://static.igem.org/mediawiki/2019/4/45/T--UCSC--day1.pdf) [Day 2](https://static.igem.org/mediawiki/2019/2/23/T--UCSC--day2.pdf) [Purification](https://static.igem.org/mediawiki/2019/d/d1/T--UCSC--purification-new.pdf)

### Notes: N/A

### Results: Successful.

![](https://static.igem.org/mediawiki/2019/7/77/T--UCSC--CAHSimg-new.png)

+ August 12 - 18

# 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.

# 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](https://static.igem.org/mediawiki/2019/e/ec/T--UCSC--ProteinPurificationProtocol.pdf)

### 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](https://static.igem.org/mediawiki/2019/0/0c/T--UCSC--zyppy.pdf) , [Transformation](https://static.igem.org/mediawiki/2019/3/3f/T--UCSC--transformation-notebook.pdf)

### 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](https://static.igem.org/mediawiki/2019/4/45/T--UCSC--day1.pdf) [Day 2](https://static.igem.org/mediawiki/2019/2/23/T--UCSC--day2.pdf) [Purification](https://static.igem.org/mediawiki/2019/d/d1/T--UCSC--purification-new.pdf)

### Notes: N/A

### Results: No Protein Produced - Failure

![](https://static.igem.org/mediawiki/2019/4/4e/T--UCSC--proteinProd.png)

+ August 19 - 25

# 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.

# 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](https://static.igem.org/mediawiki/2019/4/45/T--UCSC--day1.pdf) [Day 2](https://static.igem.org/mediawiki/2019/2/23/T--UCSC--day2.pdf) , [Purification](https://static.igem.org/mediawiki/2019/d/d1/T--UCSC--purification-new.pdf)

### 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](https://static.igem.org/mediawiki/2019/4/45/T--UCSC--day1.pdf) [Day 2](https://static.igem.org/mediawiki/2019/2/23/T--UCSC--day2.pdf) [Purification](https://static.igem.org/mediawiki/2019/d/d1/T--UCSC--purification-new.pdf)

### Notes: Improper protein production methods. → Troubleshooting

### Results: Results inconclusive again

+ August 26 - September 1

# 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

# 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](https://static.igem.org/mediawiki/2019/4/45/T--UCSC--day1.pdf) [Day 2](https://static.igem.org/mediawiki/2019/2/23/T--UCSC--day2.pdf) [Purification](https://static.igem.org/mediawiki/2019/d/d1/T--UCSC--purification-new.pdf)

### 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.

September

+ September 2 - 8

# 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

# 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](https://static.igem.org/mediawiki/2019/4/45/T--UCSC--day1.pdf) [Day 2](https://static.igem.org/mediawiki/2019/2/23/T--UCSC--day2.pdf) [Purification](https://static.igem.org/mediawiki/2019/d/d1/T--UCSC--purification-new.pdf)

### Notes: To test if primers did indeed form a hairpin and prevent transcription of the protein.

### Results: Successfully produced CAHS1.

+ September 9 - 15

# 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.

# 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](https://static.igem.org/mediawiki/2019/5/59/T--UCSC--mutagenesis.pdf) [Gel Electrophoresis](https://static.igem.org/mediawiki/2019/5/53/T--UCSC--gel-electro.pdf)

### Notes: N/A

### Results: Added a His Tag to successful plasmids, of which all had at least one.

![](https://static.igem.org/mediawiki/2019/c/c1/T--UCSC--sept9.png)

## 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](https://static.igem.org/mediawiki/2019/5/59/T--UCSC--mutagenesis.pdf) [Gel Electrophoresis](https://static.igem.org/mediawiki/2019/5/53/T--UCSC--gel-electro.pdf)

### 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](https://static.igem.org/mediawiki/2019/e/ec/T--UCSC--ProteinPurificationProtocol.pdf)

### 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.

+ September 16 - 22

# 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.

# 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](https://static.igem.org/mediawiki/2019/8/89/T--UCSC--index-plating.pdf)

### 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](https://static.igem.org/mediawiki/2019/0/0c/T--UCSC--zyppy.pdf)

### 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](https://static.igem.org/mediawiki/2019/3/3f/T--UCSC--transformation-notebook.pdf)

### 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 EggsParticipants: N/AProtocols: N/ANotes: One egg arrived crackedResults: Began incubating the eggs and turning them three times a day. Protein Production of His Tagged SAHS10(G3), LEA1(A4)-PilotParticipants: James, Melody, MartinaProtocols: [Day 1](https://static.igem.org/mediawiki/2019/4/45/T--UCSC--day1.pdf) [Day 2](https://static.igem.org/mediawiki/2019/2/23/T--UCSC--day2.pdf) [Purification](https://static.igem.org/mediawiki/2019/d/d1/T--UCSC--purification-new.pdf) Notes: Purification steps were changed to heat solubilize proteins in the autoclave instead of using a 95C water bathResults: Gels were too faded to tell if results were good or not. Started protein production again.![](https://static.igem.org/mediawiki/2019/c/cf/T--UCSC--NotebookScreenshot3.png) Protein Production of His tagged CAHSD-PilotParticipants: Varuna, Andrew, IkeProtocols: [Day 1](https://static.igem.org/mediawiki/2019/4/45/T--UCSC--day1.pdf) [Day 2](https://static.igem.org/mediawiki/2019/2/23/T--UCSC--day2.pdf) [Purification](https://static.igem.org/mediawiki/2019/d/d1/T--UCSC--purification-new.pdf) Notes: N/AResults: Successful.![](https://static.igem.org/mediawiki/2019/9/9c/T--UCSC--NotebookScreenshot4.png) SDM Second Round of CAHS1Participants: Shayan, JamesProtocols: [Site Directed Mutagenesis](https://static.igem.org/mediawiki/2019/5/59/T--UCSC--mutagenesis.pdf) [Gel Electrophoresis](https://static.igem.org/mediawiki/2019/5/53/T--UCSC--gel-electro.pdf) Notes: N/AResults: Successful. ![](https://static.igem.org/mediawiki/2019/1/13/T--UCSC--image17.png) Transformation, Index Plating, and Liquid Cultures of CAHS1(D4)Participants: James, Melody, MartinaProtocols:Notes: N/AResults: Successful. Stored to be miniprepped next week.![](https://static.igem.org/mediawiki/2019/9/9f/T--UCSC--NotebookScreenshot5.png) Analysis of SDM CAHS2 Plasmid (D3), Transformation Plates, and Index Plates by SequencingParticipants: JamesProtocols: [Site Directed Mutagenesis](https://static.igem.org/mediawiki/2019/5/59/T--UCSC--mutagenesis.pdf) [Gel Electrophoresis](https://static.igem.org/mediawiki/2019/5/53/T--UCSC--gel-electro.pdf) 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.

+ September 23 - 29

# 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.

# 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](https://static.igem.org/mediawiki/2019/5/59/T--UCSC--mutagenesis.pdf) [Gel Electrophoresis](https://static.igem.org/mediawiki/2019/5/53/T--UCSC--gel-electro.pdf)

### 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](https://static.igem.org/mediawiki/2019/4/45/T--UCSC--day1.pdf) [Day 2](https://static.igem.org/mediawiki/2019/2/23/T--UCSC--day2.pdf) [Purification](https://static.igem.org/mediawiki/2019/d/d1/T--UCSC--purification-new.pdf)

### Notes: N/A

### Results: SuccessProtein Production His TaggedCAHSD-Large ScaleParticipants: Varuna, AndrewProtocols: [Protein Production Large Scale](https://static.igem.org/mediawiki/2019/e/ec/T--UCSC--ProteinPurificationProtocol.pdf) Notes: CAHSD had a huge band!Results: Successful.![](https://static.igem.org/mediawiki/2019/2/24/T--UCSC--NotebookScreenshot6.png) Miniprep of His Tagged CAHS2(D3)Participants: James, MelodyProtocols:Notes: N/AResults: Failed. DNA yields extremely low. Attempted again same issue. Began troubleshooting.

October

+ September 30 - October 6

# 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.

# 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](https://static.igem.org/mediawiki/2019/a/a7/T--UCSC--protein-production-new.pdf)

### Notes: N/A

### Results: Successful.

![](https://static.igem.org/mediawiki/2019/b/ba/T--UCSC--NotebookScreenshot7.png)

## Large Scale Production and Purification of CAHS1(D4), LEA1(A4), SAHS10(G3)

### Participants: James, Melody, Martina

### Protocols: [Protein Production Large Scale](https://static.igem.org/mediawiki/2019/e/ec/T--UCSC--ProteinPurificationProtocol.pdf)

### Notes: Insoluble fractions were loaded with too much protein and caused the gel to streak.

### Results: Successful.

![](https://static.igem.org/mediawiki/2019/6/6b/T--UCSC--NotebookScreenshot8.png)

## 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.

![](https://static.igem.org/mediawiki/2019/4/40/T--UCSC--NotebookScreenshot9.png)

+ October 7 - 13

# 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.

# 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](https://static.igem.org/mediawiki/2019/4/45/T--UCSC--day1.pdf) [Day 2](https://static.igem.org/mediawiki/2019/2/23/T--UCSC--day2.pdf) [Purification](https://static.igem.org/mediawiki/2019/d/d1/T--UCSC--purification-new.pdf)

### 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](https://static.igem.org/mediawiki/2019/9/9b/T--UCSC--HistidinePurificationProtocol.pdf)

### 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.

![](https://static.igem.org/mediawiki/2019/a/a8/T--UCSC--NotebookScreenshot10.png)

+ October 14 - 20

# 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.

# 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](https://static.igem.org/mediawiki/2019/4/45/T--UCSC--day1.pdf) [Day 2](https://static.igem.org/mediawiki/2019/2/23/T--UCSC--day2.pdf) [Purification](https://static.igem.org/mediawiki/2019/d/d1/T--UCSC--purification-new.pdf)

### Notes: N/A

### Results: No Protein Produced.

## Plaque Assays

### Participants: Aren, Jonah, Iris

### Protocols: [Plaque Assays](https://static.igem.org/mediawiki/2019/a/af/T--UCSC--plaq-assay.pdf)

### Notes: Tried different amounts of media

### Results: Partial success. Cells were now sticking to the plate and being dyed successfully.

![](https://static.igem.org/mediawiki/2019/7/75/T--UCSC--plaque-assay.jpg)

## 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](https://static.igem.org/mediawiki/2019/9/9b/T--UCSC--HistidinePurificationProtocol.pdf)

### Notes: Gel for CAHSD broke a bit.

### Results: Success for SAHS10 and CAHSD. LEA1 failed.

![](https://static.igem.org/mediawiki/2019/4/4e/T--UCSC--proteinProd.png)
![](https://static.igem.org/mediawiki/2019/8/80/T--UCSC--NotebookScreenshot11.png)


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# Team:UCSC/Experiments

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* Team▾
  + [Team](https://2019.igem.org/Team%3AUCSC/Team)
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* Project ▾
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* Parts ▾
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* [Safety](https://2019.igem.org/Team%3AUCSC/Safety)
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  + [Entrepreneurship](https://2019.igem.org/Team%3AUCSC/Entrepreneurship)

Description Page

#### Experiments

#### Molecular Cloning

▶ LB Medium and LB/Kan plates

▶ Plasmid elution and Transformation into DH5-α

▶ Index Plating

▶ Liquid Cultures and Colony PCR

▶ TBE Buffer and Gel Production

▶ Plasmid Miniprep from DH5-α

#### Site Directed Mutagenesis

▶ Primer production and NEB Site-Directed Mutagenesis

▶ Transformation into Zymo Mix&Go DH5-α

▶ Plasmid Miniprep from DH5-α(Refer to Molecular Cloning #4)

#### Protein Production and Quantification

▶ Large Scale Protein Production and Purification

▶ Transform plasmid into XJb (DE3) Mix&Go(Refer to Site-Directed Mutagenesis #2)

▶ Start Liquid Cultures of XJb (DE3)(Refer to Molecular Cloning #3)

▶ Induction of Protein Expression via IPTG

▶ Purification via Heat-Soluble Precipitation and His-Spin Column

▶ Histidine Purification through Gravity

#### Thermostability Testing

▶ Incubation of Embryonated Chicken Eggs and Candling Procedure

▶ Red Blood Cell Preparation and Hemagglutination Assay

▶ Fibroblast Cell Culture & Plaque Assay

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# Team:UCSC/Results

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* Team▾
  + [Team](https://2019.igem.org/Team%3AUCSC/Team)
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* Project ▾
  + [Description](https://2019.igem.org/Team%3AUCSC/Description)
  + [Design](https://2019.igem.org/Team%3AUCSC/Design)
  + [Experiments](https://2019.igem.org/Team%3AUCSC/Experiments)
  + [Notebook](https://2019.igem.org/Team%3AUCSC/Notebook)
  + [Results](https://2019.igem.org/Team%3AUCSC/Results)
* Parts ▾
  + [Parts](https://2019.igem.org/Team%3AUCSC/Parts)
* [Safety](https://2019.igem.org/Team%3AUCSC/Safety)
* Human Practices ▾
  + [Integrated](https://2019.igem.org/Team%3AUCSC/Human_Practices)
  + [Engagement & Education](https://2019.igem.org/Team%3AUCSC/Public_Engagement)
  + [Entrepreneurship](https://2019.igem.org/Team%3AUCSC/Entrepreneurship)

#### Results

#### Safety

1. [Overview](#overview)
2. [Flag Tag PP](#flag)
3. [SDM](#sdm)
4. [Prot. Prod.&Purific.](#prod)
5. [HIS Column](#his)
6. [HA](#ha)
7. [Plaque Assay](#plaque)

#### Overview

We did site-directed mutagenesis to substitute our proteins containing an N-terminal FLAG-tag with a 6xHis-tag and expressed the new his-tagged proteins CAHS1(D4),CAHS2(D3), LEA1(A4), SAHS10(G3) successfully. We performed virus propagation through embryonated chicken eggs and extracted allantoic fluid containing the live, lentogenic NDV LaSota strain. We also isolated red blood cells (RBCs) from whole turkey blood successfully and ran the hemagglutination assay on our vaccine and allantoic fluid to verify that our NDV vaccine was indeed alive.

#### Flag Tag Protein Production

During the first phase of our project, we performed a large scale protein production and purification of native CAHS D. We also conducted large scale production of CAHS2 (D3), CAHS1 (D4), SAHS10(G3), LEA1(A4), and rvLEAM(H4); all five of which contained an N-terminal FLAG-tag. After we received a product, we autoclaved them to purify the proteins. The SDS-PAGE gel of the purified products shows that only CAHS D, CAHS1, SAHS 10 and LEA1 had good yield. As a result, we decided to continue site-directed mutagenesis with these four proteins.

![](https://static.igem.org/mediawiki/2019/8/87/T--UCSC--flag-tag.png)

**Figure:**
SDS-PAGE gel of purified protein from large scale production. CAHS D, CAHS 1, SAHS 10, and LEA1 have a significant band corresponding to the size of the protein produced.

Since the first step in safety is minimizing the exposure to risk, our team worked hard to eliminate risk wherever possible. We had access to a dry lab space separate from the lab where we could work on various aspects of our project that did not involve direct experimentation. This allowed us to maintain a more controlled lab space. Our team also limited the use of hazardous reagents or equipment when we had access to safer alternatives. For example, we used SYBR Safe gel stain instead of ethidium bromide so that our imaging could be done without UV.

#### Site Directed Mutagenesis

The reason we did site directed mutagenesis was to remove the FLAG tag with the His tag so that we could use the His column to purify the protein after we had finished producing it. To do this, we needed to use the NEB base changer to design primers that would replace the FLAG tag with an N-terminal 6xHis tag. SDM was then performed using a Q5 polymerase system as well as touchdown PCR based on the manufacturer protocol. Our products were ran in gels as well as sequenced to see the success of the procedure and if the His tag was implemented. Upon confirmation of the procedure, we then transformed the plasmids into dh5-α cells to have stocks of it.

![](https://static.igem.org/mediawiki/2019/c/cd/T--UCSC--sdm.png)

**Figure. Gel of results of Site Directed Mutagenesis of CAHS1(D4),CAHS2(D3), LEA1(A4),rvLEAM(H4), SAHS10(G3)**
This gel shows the results of site directed mutagenesis and the removal of the FLAG tag with a His tag. The presence of bands in the gel indicates that the site-directed mutagenesis was successful.

#### Protein Production and Purification

Protein Purification after Site Directed Mutagensis

For weeks we were trying to perform protein production with our plasmids and continued failing in producing any. We tried troubleshooting it in multiple ways but still received the same results. Upon looking at the actual structure of the sequence, we realized that we had created a hairpin terminator, which would not allow any protein to be translated.

We ran the pilot protein production of His-tagged CAHSD, CAHS1, LEA1, and SAHS 10 to verify if they were able to be expressed or not. After we verified that they got expressed from pilot expression, we proceeded to large scale protein production and purified the product through autoclaving. The SDS-PAGE gel of the purified large scale proteins indicated there were some other unknown proteins left over after autoclaving, so we did the his-column purification next for further purify.

![](https://static.igem.org/mediawiki/2019/4/4a/T--UCSC--prot-results1.png)

**Figures**
SDS-PAGE gel of purified 6xHis-tagged CAHS D from pilot expression. Bands in red boxes indicated we got purified protein in heat-soluble fraction.

![](https://static.igem.org/mediawiki/2019/8/8a/T--UCSC--prot-results2.png)

**Figures**
SDS-PAGE gel of purified 6xHis-tagged CAHS1 from pilot expression. Bands in red boxes indicated we got purified protein in heat-soluble fraction.

![](https://static.igem.org/mediawiki/2019/4/40/T--UCSC--prot-results3.png)

**Figure. SDS-PAGE gel of purified 6xHis-tagged SAHS 10 from pilot expression.**
Bands in red boxes indicated that we got purified SAHS10 in heat-soluble fraction.

![](https://static.igem.org/mediawiki/2019/3/36/T--UCSC--prot-results4.png)

**Figure. SDS-PAGE gel of purified 6xHis-tagged CAHS1(D4), CAHSD, LEA1(A4), and SAHS10(G3) from large scale production.**
Bands in red boxes indicated we got purified proteins in heat-soluble fraction.

#### HIS Column Purification

After we purified proteins by autoclaving the large scale product, we ran the his-column to further purify the His-tagged proteins. By running through different concentration of imidazole in elution buffer, we found out that 100mM is the best concentration for eluting protein.

![](https://static.igem.org/mediawiki/2019/b/b3/T--UCSC--his-results-redo.png)

**Figure. SDS-PAGE gel of His-column purified CAHS D.**
This image shows successful purification of CAHS D. Based on the results of the gel, we can say our best results occur when the samples were in 100mM elution buffer.

![](https://static.igem.org/mediawiki/2019/b/b9/T--UCSC--his-results2.png)

**Figure. SDS-PAGE gel of His-column purified CAHS 1.**
Purified CAHS1 away from DNA and the remaining proteins. The supernatant was not filtered before use, the column was clogged, making purification take over 20 hours. In addition, it appears we overloaded the column as there was still a significant amount of protein that did not bind. We could get better yield after optimized the imidazole concentration in elution buffer.

![](https://static.igem.org/mediawiki/2019/e/ee/T--UCSC--his-results1-redo.png)

**Figure. SDS-PAGE gel of His-column purified SAHS 10.**
The image displays successful purification of the protein. The band in the red box indicated that we got the most yield of protein from 100mM imidazole elution buffer.

#### Hemagglutination Assay

We got the whole Turkey blood and isolated RBCs by following the protocol  [Isolation of RBCs.](https://static.igem.org/mediawiki/2019/c/cb/T--UCSC--red-blood-results.pdf)

In order to test if we have hemagglutinating agent in the vaccine sample and if the RBCs we isolated before will auto-hemagglutinate, we did a rapid hemagglutination test and looked at the sample under the microscope. The combination of vaccine and the RBCs had clumped appearance which distincted from the control of PBS with RBCs after mixing about one minute. However, after 10 minutes heat inactivating, the vaccine sample combined with the RBCs had the same appearance with the control of PBS with RBCs. This rapid test indicated that we could use hemagglutination assay to test the infectivity of the NDV-La Sota strain in the future.

![](https://static.igem.org/mediawiki/2019/5/52/T--UCSC--pbsControl.jpg)
![](https://static.igem.org/mediawiki/2019/a/a4/T--UCSC--heat-inactivated.jpg)
![](https://static.igem.org/mediawiki/2019/d/df/T--UCSC--vaccine-results.jpg)

**Figures: From Left to Right Rapid hemagglutination test under microscope**

1.) Control of RBCs in PBS.

2.) 10 minutes heat inactivated vaccine sample with RBCs.

3.) New stock of vaccine with RBCs

After we did the rapid test and determined our isolated RBCs are ready to run the hemagglutination assay, we performed the HA assay with both new stock, old stock vaccine, allantoic fluid from both new and old vaccine stock and the 10^-5 dilution of the new vaccine stock.

![](https://static.igem.org/mediawiki/2019/7/7c/T--UCSC--hemogRes1.jpg)

**Figure. HA of the new and old stock vaccine of different dilution. Both of the new and old stock vaccine have HA titer of 128.**

![](https://static.igem.org/mediawiki/2019/0/01/T--UCSC--hemoglut2.jpg)
![](https://static.igem.org/mediawiki/2019/9/9b/T--UCSC--HA3.JPG)

**Figures: From Left to Right**

Figure. Hemagglutination assay of Allantoic fluid from embryonated eggs after virus propagation. In the left figure, the first two lanes stand for the allantoic fluid from old vaccine stock and the HA titer is 16. The following two lanes stand for the allantoic fluid from new vaccine stock and the HA titer is 32. The next two lanes stand for the allantoic fluid from -5 dilution of new vaccine stock and the HA titer is 8. In the right figure, the first two lanes stand for the allantoic fluid from 10^-3 new vaccine stock and the HA titer is 2. The following two lanes stand for the allantoic fluid from 10^-1 new vaccine stock and the HA titer is 32. The bottom two lanes of both of the figures stand for negative control which was RBCs without virus.

Our first rapid hemagglutination test of the vaccine stock under heating conditions indicated virus death when exposed to heat at 56 °C for 10 minutes. We performed more rapid hemagglutination tests under heat using different times and temperatures. Each test contained one positive control-vaccine sample with RBCs we had previously tested and verified successful hemagglutination and one negative control-PBS with RBCs which we tested to verify the RBCs will not auto-hemagglutinated. The results we looked under microscope indicated that virus under heating at 37.5℃ and 26.6℃ for 10 minutes stayed alive. Virus under heating at 56℃ for 5 minutes still died which indicated that 56℃ is a significant killing temperature to NDV-La Sota strain. We also did a test of virus centrifuged at 3,000 rcf under 37.5℃ and the result showed the virus stayed alive. This indicated that when we dilfiltrated the combination of the virus and proteins through the centrifuge, the virus stayed alive, so our HA assay on them were reliable.

![](https://static.igem.org/mediawiki/2019/6/6e/T--UCSC--final-result.png)

**Figure. Duplicated Rapid HA test under different conditions.**

![](https://static.igem.org/mediawiki/2019/8/8a/T--UCSC--hemo1.png)

**Figure. The first two lanes stand for negative control. The next two lanes stand for positive control and the next two lanes stand for 37.5 degree with TDP after 10 minutes.
and the last two lanes stand for 37.5 degrees without TDP after 10 minutes.**

![](https://static.igem.org/mediawiki/2019/8/8e/T--UCSC--real-final-result2.jpg)

**Figure. First two lanes stand for difiltration with TDP. Didn't have enough. smaller than 50 ul spun without TDP**

#### Plaque Assay

Our plaque assay tests were failed for the first two rounds. Thus, we spent two weeks diagnosing our procedure. We finally found out that the concentration of the agar is the essential problem here. By running the 0.9%, 0.6% and 0.3% agar overlay medium with both 2X PBS and 2X TPB on the same condition of chicken fibroblast cell monolayer, we figured out the 0.3% agar

![](https://static.igem.org/mediawiki/2019/5/50/T--UCSC--plaq-assay1.jpg)
![](https://static.igem.org/mediawiki/2019/d/de/T--UCSC--plaq-assay2.jpg)

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