iGEM 2022 — Team ucsc

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[June](#June)
[July](#July)
[August](#August)
[September](#September)
[October](#October)
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### **June**

## Week of 6/27/2022

1. **Wiki**
:   - Brainstormed organization and illustrated design of wiki layout on whiteboard in dry lab
:   - Learned and practiced languages for wiki design using W3schools and other resources as needed
2. **Human Outreach**
:   To date: have met with endocrinologists, nephrologists, medical doctors, directors of health centers, and diabetes support program coordinators.

    Dr. Rayhan Lal (5/14/22), Doctors Without Borders (5/17/22), Dr. Patricia Wu (6/22/22), Dr. John Demko (6/23/22), Dr. Sunder Mudaliar (6/24/22), Dr. Lawrence DeGhetaldi (6/29/22), Dr. Matt Sparke (6/30/22) provided thorough insight for current Type II Diabetes treatment that shaped project development preparation
3. **Host Organisms**
***Yeast:***:   - Researched yeast and microbial strains, [bioencapsulation](https://2022.igem.wiki/ucsc/glossary#Bioencapsulation) methods, [half-life](https://2022.igem.wiki/ucsc/glossary#half-life) extenders, [Ex-4](https://2022.igem.wiki/ucsc/glossary#Ex-4 "Exendin-4") functionality, [plasmids](https://2022.igem.wiki/ucsc/glossary#Plasmid) for select strains
:   - Looked into strains and backbones for our experiment:   :   - Met with Prof. Rohinton Kamakaka at UCSC for yeast protein synthesis and received yeast strain and plasmid backbone from his lab
        ***Microalgae:***:   - Completed a microalgae protocol, timeline, list of materials, and line itemized cost list
        - **Plasmid Design**
        :   - Constructed Ex-4 insert in pET28:GFP and yeast backone plasmids
        :   - Ordered plasmid parts from IDT

---

### **July**

## Week of 7/4/2022

1. **Wiki**
:   - Coded in IDE for wiki pages
:   - Brainstormed for theme and aesthetics
2. **Human Outreach**
:   - Admin team: sent thank you emails for giving month
:   - Prepared investment portfolio; meeting with Paul L. Koch, PBSci Dean at UCSC
:   - connected with iGEM teams for collaboration (Chihuahua, Mexico and Stoneybrook, New York)
:   - Met with Dr. Somen Nandi (7/5/22), from UC Davis, for project development input and Ted Malpass (7/6/22), from UCSC, who has Type 1 Diabetes and works at a nonprofit for accesible medical treatment
3. **Host Organisms**
***Yeast:***:   - Finalized new [plasmid](https://2022.igem.wiki/ucsc/glossary#Plasmid) design for available materials and ordered insert gene block
:   - Checked lab stock, developed protocols, timelined for experimental work, and conceptualized [bioencapsulation](https://2022.igem.wiki/ucsc/glossary#Bioencapsulation) methods
***Microalgae:***:   - Determined feasibility of microalgae strains from research: comparison of strains (chose *Chlamydomonas reinhardtii*), methodology of transformation (chose [AMT](https://2022.igem.wiki/ucsc/glossary#AMT)), and target location (chose nuclear genome)
:   - Prepared protocols for various selections of the above criteria
4. **Plasmid Design**
:   - Gave Golden Gate Assembly ([GGA](https://2022.igem.wiki/ucsc/glossary#GGA)) lectures to establish a collective understanding within the team
:   - Finished GGA design and ordered primers
:   - Drafted general protocols for chemicompetent cell transformation, plasmid [miniprep](https://2022.igem.wiki/ucsc/protocols#collapse8eight8), colony touchdown [PCR](https://2022.igem.wiki/ucsc/glossary#PCR "Polymerase Chain Reaction"), protein purification using a HisPur™ Cobalt Purification Kit (ThermoFisher), and HisPur Cobalt column sterilization for reuse
:   - Discussed [GLP-1 R](https://2022.igem.wiki/ucsc/glossary#GLP-1-R "Glucagon-Like Peptide 1 Receptor ") design attributes, size, cost, human vs. rat, post translational modifications, understanding and verifying GLP-1 R interaction to [Ex-4](https://2022.igem.wiki/ucsc/glossary#Ex-4 "Exendin-4")

## Week of 7/11/2022

1. **Human Outreach**
:   - Finalized iGEM project track selection (7/18/22)
:   - Met with Alexander Wolf, Baskin Engineering Dean at UCSC, for story approval
2. **Host Organisms**
***Yeast:***:   - Prepared LB and SOC media
:   - Lab preparation for [*E. coli*](https://2022.igem.wiki/ucsc/glossary#E_coli "E. coli") and [*S. cerevisiae*](https://2022.igem.wiki/ucsc/glossary#S_cerevisiae "His-tagged Tobacco Etch Virus Protease") transformations, including taking inventory
3. **Plasmid Design**
:   - Nicholas Lorig-Roach, member of DuBois Lab at UCSC, provided cells with His-[TEV](https://2022.igem.wiki/ucsc/glossary#TEV-protease "His-tagged Tobacco Etch Virus Protease") protease and cells for producing His-TEV protease
:   - Cells were cultured, given [IPTG](https://2022.igem.wiki/ucsc/glossary#IPTG "Isopropyl-&beta-D-1-thiogalactopyranoside"), and pelleted for [cell lysing](https://2022.igem.wiki/ucsc/protocols#collapse12twelve12)
:   - Lysis performed using glass beads and yield extracted through Histidine protein purification protocol by Gravity
:   - Given His-TEV protease cells were used for protein purification practice
:   - Results were inconclusive

## Week of 7/18/2022

1. **Wiki**
:   - Implemented 'sticky' navbar and fancy hyperlink underlines- **Human Outreach**
    :   - Completed first set of website commits of various elements: sticky navbar, fancy hyperlinks mk.1
    :   - Used bootstrap scrollspy with a benzene ring design
    :   - Formatted footer on wiki page
    :   - Chose team name
    - **Computational Modeling**
    :   - Start of computational modeling
    :   - Met with SBU for mathematical protein modeling and explained basics of MATLAB
    - **Host Organisms**
    ***Yeast:***:   - Practiced [*E. coli*](https://2022.igem.wiki/ucsc/glossary#E_coli "E. coli") transformation and [plasmid miniprep](https://2022.igem.wiki/ucsc/protocols#collapse8eight8) to test [plasmid](https://2022.igem.wiki/ucsc/glossary#Plasmid)
    :   - Prepared Gibson Assembly protocol
    :   - Performed *E. coli* transformation for plasmid part cloning (materials provided by Rohinton Kamakaka lab: plasmid gal1 [promoter](https://2022.igem.wiki/ucsc/glossary#Promoter), terminator, LEU- non integrative, [G418](https://2022.igem.wiki/ucsc/glossary#G418) LEU- integrative, Hyg [TRP1](https://2022.igem.wiki/ucsc/glossary#TRP1) integrative)

## Week of 7/25/2022

1. **Human Outreach**
:   - Created google form for weekly subgroup updates
:   - Researched 3D modeling display, but shifted focus to more crucial elements for the website design
:   - Reformatted google form for weekly submissions
2. **Computational Modeling**
:   - Introduction to using SimBiology
:   - Researched how [ligand](https://2022.igem.wiki/ucsc/glossary#Ligand)-[receptor](https://2022.igem.wiki/ucsc/glossary#Receptor) binding functions in depth and how [Ex-4](https://2022.igem.wiki/ucsc/glossary#Ex-4 "Exendin-4") maintains homeostasis in the body
3. **Host Organisms**
***Yeast:***:   - Prepared LB and SOC media
:   - Lab preparation for [*E. coli*](https://2022.igem.wiki/ucsc/glossary#E_coli "E. coli") and [*S. cerevisiae*](https://2022.igem.wiki/ucsc/glossary#S_cerevisiae "S. cerevisiae") transformations, including taking inventory
4. **Interlab**
:   - Completed [Calibration Experiment](https://old.igem.org/wiki/images/a/a4/InterLab_2022_-_Calibration_Protocol_v2.pdf)
:   - Completed [Experiment 1](https://old.igem.org/wiki/images/f/f8/InterLab_2022_Exp1.pdf)

---

### **August**

## Week of 8/1/2022

1. **Wiki**
:   - Worked on creating and implementing SVGs into the website: included a loading screen and worked on resizing SVG elements for compatibility with end user's screen space
2. **Computational Modeling**
:   - Researched and worked on schematics for [ligand](https://2022.igem.wiki/ucsc/glossary#Ligand)-[receptor](https://2022.igem.wiki/ucsc/glossary#Receptor) binding, [GLP-1 R](https://2022.igem.wiki/ucsc/glossary#GLP-1-R "Glucagon-Like Peptide 1 Receptor ") binding downstream effects, and recombinant protein production rates in both hosts
:   - Created a schematic for downstream insulin production in pancreatic beta cells, a mathematical model for ligand-receptor binding, and a graph for pharmacological effect vs. dosage
:   - Members of the modeling team began familiarizing themselves with the software and conducted background research.
3. **Human Outreach**
:   - Correspondence with Karianne Terry (8/5/22) for safety guidelines
:   - IRB research to ensure project safety
:   - Emailed various affected communities
:   - Met with Dr. Tércio Rocha (8/5/22) who provided perspective on the health care system in Brazil
4. **Host Organisms**
***Yeast:***:   - Innoculated LEU+ yeast strain ([ROY6783](https://2022.igem.wiki/ucsc/glossary#ROY-6783)) and LEU- yeast strain (JRY4012) in 10ml [YPD](https://2022.igem.wiki/ucsc/glossary#YPD) media, incubated overnight at 30 Celsius on shaker, lid loose
:   - Prepared selection plates for [*S. cerevisiae* transformation](https://2022.igem.wiki/ucsc/protocols#collapse11eleven11)
:   - Performed cleavage using manufacture protocol for Thermofisher FastDigest NotI Restriction Enzyme
:   - Yielded unsuccessful DNA precipitation
:   - Performed [yeast transformation](https://2022.igem.wiki/ucsc/protocols#collapseFive) with [miniprep](https://2022.igem.wiki/ucsc/protocols#collapse8eight8) [plasmid](https://2022.igem.wiki/ucsc/glossary#Plasmid) product using the ZYMO Yeast Transformation Kit
:   - Transformed non-integrative plasmids (1-1, 1-3) in LEU- yeast strain (JRY4012)
:   - Transformed [G418](https://2022.igem.wiki/ucsc/glossary#G418) [LEU2](https://2022.igem.wiki/ucsc/glossary#LEU2) integrative plasmids (2-1, 2-2) in LEU+ yeast strain (ROY6783), plated on G418 antibiotic plates
:   - Repeated transformation for successful enzyme digest
:   - Transformed Hyg-[TRP1](https://2022.igem.wiki/ucsc/glossary#TRP1) integrative plasmids (3-2, 3-4) in LEU+ yeast strain (ROY6783), plated on Hygromycin B antibiotic plates
:   - Replica plating
5. **Interlab**
:   - Started [Experiment 2](https://old.igem.org/wiki/images/f/ff/InterLab_2022_Exp2.pdf)

## Week of 8/8/2022

1. **Wiki**
:   - Learned microformatting for SEO for viewers to readily find our website
2. **Host Organisms**
***Yeast:***:   - Obtained [LEU2](https://2022.igem.wiki/ucsc/glossary#LEU2) Cas9 [plasmid](https://2022.igem.wiki/ucsc/glossary#Plasmid) from Prof. Rohinton Kamakaka
:   - Innoculated *S. cerevisiae* strains (LEU+ [ROY6783](https://2022.igem.wiki/ucsc/glossary#ROY-6783) and LEU- JRY4012) in 10ml culture 25℃ overnight
:   - Transformed *S. cerevisiae* strains
:   - Made protein expression media
:   - Replica plating
:   - Ran gel of [colony PCR](https://2022.igem.wiki/ucsc/glossary#Colony-PCR) product from [*S. cerevisiae* transformation](https://2022.igem.wiki/ucsc/protocols#collapse11eleven11)
*****E. coli****:*:   - Transformed pET28:GFP into TOP10
:   - Inverse PCR, gel, clear band showing linearized backbone
:   - [Dpn1 + heat treatment](https://2022.igem.wiki/ucsc/protocols#collapseFour) on some of the iPCR rxns, [Golden Gate Assembly](https://2022.igem.wiki/ucsc/protocols#collapseFive), [pET28:Ex-4](https://2022.igem.wiki/ucsc/glossary#pET28:Ex-4) transformed into [DH10B](https://2022.igem.wiki/ucsc/glossary#DH10B) (8/9/22):   - Transformed more pET28:Ex-4 into DH10B
    :   - Inoculated DH10B, performed colony [PCR](https://2022.igem.wiki/ucsc/glossary#PCR "Polymerase Chain Reaction"), index plates, and gels: had successful yield of [Ex-4](https://2022.igem.wiki/ucsc/glossary#Ex-4 "Exendin-4") bands:   - [Miniprep](https://2022.igem.wiki/ucsc/protocols#collapse8eight8) on innoculated pET28:Ex-4, sent for [Sanger Sequencing](https://2022.igem.wiki/ucsc/glossary#Sanger-Sequencing)
        :   - Received successful sequencing results
        - **Interlab**
        :   - Completed Experiment 2

## Week of 8/15/2022

1. **Wiki**
:   - Reorganized website layout and delegated pages with iGEM criteria to subgroups
:   - Wiki development: touched up jump to top button, fixed banner carousel at top of page, started implementing the loading screen
2. **Host Organisms**
***Yeast:***:   - Innoculated *S. cerevisiae* strains (LEU+ [ROY6783](https://2022.igem.wiki/ucsc/glossary#ROY-6783) and LEU- JRY4012) in 5mL culture for protein purification
:   - [Plasmid miniprep](https://2022.igem.wiki/ucsc/glossary#collapse8eight8)
:   - [PCR](https://2022.igem.wiki/ucsc/glossary#PCR "Polymerase Chain Reaction")
:   - [Genomic DNA isolation](collapse15fifteen15)
:   - Performed [gel electrophoresis](https://2022.igem.wiki/ucsc/protocols#collapseX) on Genomic DNA to check for integration of [Ex-4](https://2022.igem.wiki/ucsc/glossary#Ex-4 "Exendin-4") at homology sites, protein purification, and sent for [Sanger Sequencing](https://2022.igem.wiki/ucsc/glossary#Sanger-Sequencing) of Genomic DNA to check for integration of Ex-4 at homology sites
:   - Lab preparation for *E. coli* and *S. cerevisiae* transformations, including taking inventory
*****E. coli****:*:   - Transformed [pET28:Ex-4](https://2022.igem.wiki/ucsc/glossary#pET28:Ex-4) into [BL21](https://2022.igem.wiki/ucsc/glossary#BL21), innoculated transformants, index plate, and [colony PCR](https://2022.igem.wiki/ucsc/glossary#Colony-PCR)/gel
:   - Ran an agarose gel on PCR samples of transformed pET28:Ex-4 in BL21 and successful yield of Ex-4 bands
:   - Miniprep on innoculated pET28:Ex-4 and sent for Sanger Sequencing
:   - Received confirmation from sequencing that pET28:Ex-4 was successfully transfected into the *E. coli*

## Week of 8/22/2022

1. **Wiki**
:   - Notebook page development and testing; developed an SVG design for the page

## Week of 8/29/2022

1. **Wiki**
:   - Continued coding and testing various elements for notebook page (buttons and collapsibles):   - Various redesigns of notebook page format
    - **Human Outreach**
    :   - Met with NGO founder Annette Oshin (9/1/22)
    :   - promo video submitted (9/2/22)
    - **Computational Modeling**
    :   - Devised a detailed schedule for modeling work due by wiki freeze
    :   - Identified literature values for protein production rate constants, derived formulas used in the competitive binding model, coded both models
    - **Host Organisms**
    ***Yeast:***:   - *S. cerevisiae* culture innoculation for second round of protein purification

---

### **September**

## Week of 9/5/2022

1. **Wiki**
:   - A hexagonal grid using flexboxes was achieved for the Team/Attributions page
2. **Computational Modeling**
:   - Development of competitive binding model: researched parameter values, derived fractional occupancy equation, and coded the model in Python
:   - Eric Malekos helped troubleshoot our code
3. **Human Outreach**
:   - Follow up with Dr. Rayhan Lal (9/7/22)
4. **Host Organisms**
***Yeast:***:   - Performed protein purification
:   - Successful [SDS-PAGE](https://2022.igem.wiki/ucsc/protocols#collapse14fourteen14) gel showing expression of [His-tag](https://2022.igem.wiki/ucsc/glossary#His-Tag) [Golden Gate Assembly](https://2022.igem.wiki/ucsc/protocols#collapse10ten10) protein from genomic DNA
*****E. coli****:*:   - Transformed [Ex-4](https://2022.igem.wiki/ucsc/glossary#Ex-4 "Exendin-4") again into [C41](https://2022.igem.wiki/ucsc/glossary#C41) [*E. coli*](https://2022.igem.wiki/ucsc/glossary#collapse6Six6) since it has been in freezer for a long time
:   - Used freshly purified Ex-4 for our pancreatic beta cell test, [binding affinity](https://2022.igem.wiki/ucsc/glossary#Binding-Affinity) test, and Western Blot
:   - Transformation was successful and colonies were innoculated
:   - Colony [PCR](https://2022.igem.wiki/ucsc/glossary#PCR "Polymerase Chain Reaction") gel was performed
:   - Expected bands were shown proving that Ex-4 was successfully transfected into our protein producing C41 cells

## Week of 9/12/2022

1. **Wiki**
:   - Worked on the color pallet, structure of the nav bar, and reorganized layout of the wiki
:   - The flask jinja template documentation was utilized to achieve some reorganization
:   - Began organization of content for wiki pages
2. **Computational Model**
:   - Worked on the dose-effect model: Searched for parameter values, derived Emax equation, and coded the model in Python
:   - Eric Malekos viewed our graphs and provided feedback for optimization
:   - Slight changes were made to the previous competitive binding model (developed week of 9/5/2022) to better format information
3. **Human Outreach**
:   - Safety form submitted (9/16/22)
4. **Host Organisms**
*****E. coli****:*:   - Performed protein purification using an [FPLC](https://2022.igem.wiki/ucsc/glossary#FPLC "Fast Protein Liquid Chromatography") machine, but to our dismay, the FPLC needs maintenance
:   - Performed [SDS-PAGE](https://2022.igem.wiki/ucsc/glossary#SDS-PAGE "Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis") to verify if [GLP-1R](https://2022.igem.wiki/ucsc/glossary#GLP-1-R "Glucagon-Like Peptide 1 Receptor ") was purified, despite our faulty equipment
:   - Our SDS-PAGE test to confirm the FPLC purification displayed that [Ex-4](https://2022.igem.wiki/ucsc/glossary#Ex-4 "Exendin-4") was not purified
:   - Performed [IMAC](https://2022.igem.wiki/ucsc/protocols#collapse13thirteen13) protein purification by gravity
:   - Ex-4 was grown in LB/Kan and innoculated with [IPTG](https://2022.igem.wiki/ucsc/glossary#IPTG "Isopropyl-&beta-D-1-thiogalactopyranoside") to prepare for protein purification

## Week of 9/19/2022

1. **Wiki**
:   - Made miscellaneous shifts in webpage architecture to make implementation for page content more intuitive
2. **Computational Model**
:   - Worked on the protein production model: Searched for parameter values, drew up model in SimBiology, edited reaction equations for result concentration diagram
:   - Drafted a plan for modeling work to complete during Fall quarter
3. **Human Outreach**
:   - Parts page submitted (9/22/22)
4. **Host Organisms**
***Yeast:***:   - Innoculated Int-TRP and Int-LEU in 5mL of [YPD](https://2022.igem.wiki/ucsc/glossary#YPD "Yeast Extract-Peptone-Dextrose") each
:   - Incubated innoculated solutions at 30℃ for overnight growth
:   - Innoculated each of the 5mL cultures into 500 mL of YPG respectively to activate gal1 [promoter](https://2022.igem.wiki/ucsc/glossary#Promoter)
:   - Incubated innoculated solutions at 30℃ for overnight growth or until 2 [OD](https://2022.igem.wiki/ucsc/glossary#OD "Optical Density")
****E. coli*:***:   - Pelleted cells and stored in 4℃ until following day
:   - Resuspended and lysed cells (for both [C41](https://2022.igem.wiki/ucsc/glossary#C41) and [BL21](https://2022.igem.wiki/ucsc/glossary#BL21))
:   - Performed [IMAC](https://2022.igem.wiki/ucsc/glossary#IMAC "Immobilized Metal-Affinity Chromatography") protein purification of protein samples
:   - [SDS-PAGE](https://2022.igem.wiki/ucsc/glossary#SDS-PAGE "Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis") gel of purified protein

## Week of 9/26/2022

1. **Wiki**
:   - Finalized website color scheme and most wiki page content
2. **Computational Model**
:   - Continued to look for appropriate parameters for the protein production model; difficult to find ribosome binding kinetics
:   - Shifted to computational modeling wiki page development
3. **Human Outreach**
:   - Title and Abstract submitted (9/30/22)
4. **Host Organisms**
***Yeast:***:   - Pelleted cells and stored in 4℃ until following day
:   - Resuspended and lysed cells (for both LEU and TRP)
:   - Performed [IMAC](https://2022.igem.wiki/ucsc/glossary#IMAC "Immobilized Metal-Affinity Chromatography") protein purification of protein samples
:   - [SDS-PAGE](https://2022.igem.wiki/ucsc/glossary#SDS-PAGE "Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis") gel of purified protein

---

### **October**

## Week of 10/3/2022

1. **Wiki**
:   - Integration of content
:   - Created classes for consistency in page structure
:   - Style redesign; added column artwork for content pages
2. **Computational Model**
:   - Finished the protein production model by circumventing ribosome binding, assuming any ribosome that can bind to mRNA will, at a rate of 1 per second
:   - Sent protein production model to Stony Brook University to show how their recombinant protein would act in yeast
:   - Continued to develop computational model wiki page

## Week of 10/10/2022

1. **Wiki**
:   - Full team meeting to finalize wiki content
2. **Computational Model**
:   - Finished uploading computational modeling figures:   - - **Host Organisms**
          ****E. coli*:***:   - Diafiltration (on previously purified *E. coli* and *S. cerevisiae* [Ex-4](https://2022.igem.wiki/ucsc/glossary#Ex-4 "Exendin-4")) to swap elution buffer with Tris-HCL
          :   - Add [enterokinase](https://2022.igem.wiki/ucsc/glossary#Enterokinase) to those samples and incubate overnight to cut off [His-tag](https://2022.igem.wiki/ucsc/glossary#His-Tag) on Ex-4
          :   - Packed new [IMAC](https://2022.igem.wiki/ucsc/glossary#IMAC "Immobilized Metal-Affinity Chromatography") columns
          :   - Protein purification for [TRP1](https://2022.igem.wiki/ucsc/glossary#TRP1), [LEU2](https://2022.igem.wiki/ucsc/glossary#LEU2), [BL21](https://2022.igem.wiki/ucsc/glossary#BL21), and [C41](https://2022.igem.wiki/ucsc/glossary#C41)
          :   - Batch purification on enterokinase samples was unsuccessful
          :   - [SDS-PAGE](https://2022.igem.wiki/ucsc/glossary#SDS-PAGE "Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis") of new protein purification samples from *S. cerevisiae* and *E. coli*
          :   - Nothing showed up on SDS-PAGE so we ran a new SDS-PAGE with other samples from protein purification on Wednesday
          :   - [Octet assay](https://2022.igem.wiki/ucsc/glossary#Octet-Machine) on previously purified *E. coli* Ex-4 and purchased [GLP-1 R](https://2022.igem.wiki/ucsc/glossary#GLP-1-R "Glucagon-Like Peptide 1 Receptor")

{% endblock %}

{% extends "layout.html" %}
{% block head %}{% endblock %}
{% block title%}Engineering Success{% endblock %}
{% block img0url %}https://static.igem.wiki/teams/4190/wiki/banner-gif/ezgif-1-1019c9da57.gif{% endblock %}
{% block img0desc %}{% endblock %}
{% block header %}

![{{ self.img0desc() }}]({{ self.img0url() }})

# {{ self.title() }}

#### {{ self.img0desc() }}

{% endblock %}
{% block sideBarEntries %}
[Ex-4 in *E. coli*](#engineering1)
[Ex-4 in *S. cerevisiae*](#engineering2)
[Producing GLP-1 R in *E. coli*](#engineering3)
[Binding Affinity Assay](#engineering4)
[Modeling](#engineering5)
[Citations](#Citation-Accordion)
{% endblock %}
{% block page\_content %}

#### **Ex-4 in *E. coli***

##### **First Iteration**

**Design:** We derived our backbone from the pET28:GFP [plasmid](https://2022.igem.wiki/ucsc/glossary#Plasmid "Plasmid") via inverse [PCR](https://2022.igem.wiki/ucsc/glossary#PCR "PCR") [[1](#Citation-Accordion)]. pET28:GFP contains a prokaryotic origin of replication, kanamycin antibiotic resistance, green fluorescent protein (GFP), a T7 [promoter](https://2022.igem.wiki/ucsc/glossary#Promoter "Promoter") and a T7 terminator. The [*E. coli*](https://2022.igem.wiki/ucsc/glossary#E_coli "E_coli") codon optimized [Ex-4](https://2022.igem.wiki/ucsc/glossary#Ex-4 "Ex-4") sequence was inserted into the space occupied by GFP on the pET28:GFP plasmid. Our Ex-4 gene insert includes: BsaI restriction enzyme site, [Shine-Dalgarno](https://2022.igem.wiki/ucsc/glossary#Shine-Dalgarno "Shine-Dalgarno") sequence, [His-Tag](https://2022.igem.wiki/ucsc/glossary#His-Tag "His-Tag"), linker sequence, [enterokinase](https://2022.igem.wiki/ucsc/glossary#Enterokinase "Enterokinase") [cut site](https://2022.igem.wiki/ucsc/glossary#Cut-site "Cut-site"), and the Ex-4 codon optimized sequence. To learn more about each of the components of our Ex-4 gene insert, check out our [Contributions](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27contribution%27%29%20%7D%7D) page!

**Build:** Golden Gate Assembly ([GGA](https://2022.igem.wiki/ucsc/glossary#GGA "GGA")) was used to insert Ex-4 into the plasmid backbone. GGA is a modular assembly method by which multiple DNA fragments can be simultaneously integrated into a plasmid backbone [[2](#Citation-Accordion)]. GGA exploits type IIS restriction enzymes, which cleave DNA surrounding
the recognition site, so the final [recombinant](https://2022.igem.wiki/ucsc/glossary#Recombination "Recombination") product has no indication of integration. All type II restriction enzymes leave [sticky ends](https://2022.igem.wiki/ucsc/glossary#Sticky-Ends "Sticky-Ends") on the backbone and the gene insert. These sticky ends bind to one another since they are designed to have complementary nucleotide sequences. Specifically, type IIS restriction enzymes cut downstream of the binding site by a certain number of nonspecific nucleotides. This number varies based on the type IIS restriction enzyme used. This means the sticky ends can be engineered to seamlessly integrate DNA without inserting residual nucleotides from the restriction enzyme binding site. This is deemed “scarless integration”, and prevents [mutagenesis](https://2022.igem.wiki/ucsc/glossary#Mutagenesis "Mutagenesis") during transcription of the amino acid sequences. The deletion of restriction enzyme sites also means digestion and ligation can be accomplished simultaneously.

**Test:** Once Ex-4 containing plasmids were constructed via a one step digestion and ligation GGA, they were transformed into [DH10B](https://2022.igem.wiki/ucsc/glossary#DH10B "DH10B") *E. coli*. No colonies grew after this first GGA attempt.

##### **Second Iteration**

**Design & Build:** Consequently, we decided to try a longer thermocycler protocol that cycles through digestion and ligation steps to increase the probability of successful plasmid construction. For this next attempt using GGA, we pursued both the shorter and longer [protocols](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocols%27%29%20%7D%7D).

**Test & Learn:** These plasmids were again transformed into DH10B *E. coli* and incubated overnight. Colonies grew after this transformation, implying the longer thermocycler protocol may have contributed to a higher transformation efficiency. After testing both settings, we decided to use the longer thermocycler setting during GGA for constructing our [pET28:Ex-4](https://2022.igem.wiki/ucsc/glossary#pET28:Ex-4 "pET28:Ex-4") plasmid. After we performed [colony PCR](https://2022.igem.wiki/ucsc/glossary#Colony-PCR "Colony-PCR") on the DH10B transformants and performed gel electrophoresis, we sent samples with bands at the expected size to sequencing. Of the seven samples sent to sequencing, only one contained a mutation. Plasmid samples with our Ex-4 inserts displaying no mutations in sequencing results were then transformed into the protein producing *E. coli* [BL21](https://2022.igem.wiki/ucsc/glossary#BL21 "BL21") and [C41](https://2022.igem.wiki/ucsc/glossary#C41 "C41").

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#### **Ex-4 in *S. cerevisiae***

##### **First Iteration**

**Design:** For the construction of our *S. cerevisiae* genomic insert, we used a [GAL1 promoter](https://2022.igem.wiki/ucsc/glossary#GAL1-promoter "GAL1-promoter") and a [PGK1](https://2022.igem.wiki/ucsc/glossary#PGK1 "PGK1") terminator. The GAL1 promoter and PGK1 terminator sequences are derived from the yeast Modular Cloning ([MoClo](https://2022.igem.wiki/ucsc/glossary#MoClo "MoClo")) library [[3](#Citation-Accordion)]. Protein production is initiated by the GAL1 promoter when *S. cerevisiae* is grown up in a galactose based liquid media.

We used the plasmid backbones [mg-int-leu2-kan-1420](https://2022.igem.wiki/ucsc/glossary#mg-int-leu2-kan-1420 "mg-int-leu2-kan-1420") (hereafter referred to as [pLEU](https://2022.igem.wiki/ucsc/glossary#pLEU "pLEU")) and [mg-int-trp1-hyg-1432-4a](https://2022.igem.wiki/ucsc/glossary#mg-int-trp1-hyg-1432-4a "mg-int-trp1-hyg-1432-4a") (hereafter referred to as [pTRP](https://2022.igem.wiki/ucsc/glossary#pTRP "pTRP")) for eventual genomic integration of our Ex-4 gene insert. pLEU has an origin of replication for *E. coli*, a yeast [G418](https://2022.igem.wiki/ucsc/glossary#G418 "G418") resistance as a selection marker and [LEU2](https://2022.igem.wiki/ucsc/glossary#LEU2 "LEU2") homology sites for integration into the host genome. pTRP is identical to the previous plasmid, except that it has hygromycin resistance in place of G418 resistance and [TRP1](https://2022.igem.wiki/ucsc/glossary#TRP1 "TRP1") homology sites in place of LEU2. This means that this plasmid is integrated at a different spot inside the genome. Our insert was placed in between the two BsaI cut sites [[4](#Citation-Accordion)] already present on both the plasmid backbones. Because the promoter and terminator were cut out by our restriction enzyme digest, we added these parts as well during GGA.

The *S. cerevisiae* Ex-4 sequence was optimized through IDT [[5](#Citation-Accordion)]. An elastin-based polypeptide half-life extender sequence was added to our insert on the C-terminus. The half-life extender is composed of a ten-fold repeat of the VPGVG amino acid sequence, and increases the half-life of Ex-4 by 3.7 times [[6](#Citation-Accordion)]. We hypothesize this may allow for increased [bioavailability](https://2022.igem.wiki/ucsc/glossary#Bioavailability "Bioavailability") of orally administered Ex-4. To learn more about each of the components of the *S. cerevisiae* genomic insert, check out our [Contributions](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27contribution%27%29%20%7D%7D) page!

**Build:** Our genomic insert for *S. cerevisiae* was assembled using the yeast MoClo Library [[3](#Citation-Accordion)]. This pivotal research in yeast enables rapid design of plasmids for protein expression. The MoClo library provides resources for easy, one-step constructions of yeast plasmids through GGA. Predesigned plasmid parts can be interchanged quickly, allowing for easy testing of multiple plasmid constructs.

The plasmid backbones, GAL1 promoter, Ex-4 insert, and PGK1 terminator were assembled simultaneously through a one tube GGA reaction. These customized plasmids were subsequently grown in DH10B *E. coli* post GGA and then introduced into our yeast strains. We transformed [pLEU:Ex-4](https://2022.igem.wiki/ucsc/glossary#pLEU:Ex-4 "pLEU:Ex-4") into *S. cerevisiae* strain number [ROY 6783](https://2022.igem.wiki/ucsc/glossary#ROY-6783 "ROY-6783"). ROY 6783 has a wild type LEU2 gene in the chromosome, allowing integration and [auxotrophic](https://2022.igem.wiki/ucsc/glossary#Auxotrophic "Auxotrophic") selection within the leucine production site of the genome. For the [pTRP:Ex-4](https://2022.igem.wiki/ucsc/glossary#pTRP:Ex-4 "pTRP:Ex-4") plasmid, we utilized *S. cerevisiae* strain number [ROY 4513](https://2022.igem.wiki/ucsc/glossary#ROY-4513 "ROY-4513"). ROY 4513 has a wild type TRP1 gene in the chromosome, allowing integration and auxotrophic selection within the tryptophan production site of the genome.

**Testing:** We performed transformation of *S. cerevisiae* with the linearized integrative plasmids purified from our DH10B *E. coli* colonies. An additional linear plasmid was utilized in the pLEU:Ex4 transformation culture. This plasmid contained the [CRISPR-Cas9](https://2022.igem.wiki/ucsc/glossary#CRISPR-Cas9 "CRISPR-Cas9") protein coding gene and a single guide RNA targeting the LEU2 integration sites. The CRISPR-Cas9 protein cleaves and creates a chromosome break on the integration sites, which promotes [homologous repair](https://2022.igem.wiki/ucsc/glossary#Homologous-Repair "Homologous-Repair") and increases the efficiency of integration of the pLEU plasmid.

The genomic DNA samples from pLEU:Ex4 and pTRP:Ex4 transformants were used for PCR reactions to check for gene insert integration via amplification. For each template DNA, the forward primer on the GAL1 promoter and the reverse primer on the PGK1 terminator were used to amplify the complete sequence of Ex-4. Bands at the expected size confirmed the presence of the desired protein coding sequence. Another set of primer pairs designed around the LEU2 [homology region](https://2022.igem.wiki/ucsc/glossary#Homology-region "Homology-region") and TRP1 homology region were run in separate PCR reaction mixtures to amplify the wildtype and integrated sequence of LEU2 and TRP1 integration sites. Lastly, the primer pair used previously to confirm GGA before the *S. cerevisiae* transformation step was used as a positive control.

PCR products of the Ex-4 [amplicon](https://2022.igem.wiki/ucsc/glossary#Amplicon "Amplicon") from the *S. cerevisiae* genome were prepared and sent for single primer extension [Sanger Sequencing](https://2022.igem.wiki/ucsc/glossary#Sanger-Sequencing "Sanger-Sequencing"). The same forward primer and reverse primer used for PCR reactions were used for separate sequencing on the forward and reverse strands of each sample. Lastly, we lysed our cells to extract Ex-4 and purified it through immobilized metal affinity chromatography ([IMAC](https://2022.igem.wiki/ucsc/glossary#IMAC "IMAC")). We ran our purified protein on an [SDS-PAGE](https://2022.igem.wiki/ucsc/glossary#SDS-PAGE "SDS-PAGE") to see if there was a His-tag protein at the expected size of our Ex-4 protein.

**Learn:** Our genomic DNA gel electrophoresis showed bands at the expected nucleotide size for all sets of primers at both the LEU2 and TRP1 sites. To confirm that these nucleotide segments matched what we digitally designed, we sent our PCR products for Sanger Sequencing. We observed a 100% match on the Ex-4 protein coding region from all four sequencing results. Our SDS-PAGE results further confirmed that there was a His-tag protein of the appropriate size being produced by our yeast. Our work with *S. cerevisiae* also taught our team how to successfully perform a genomic integration of DNA into *>S. cerevisiae*.

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#### **GLP-1 R in *E. coli***

##### **First Iteration**

**Design:** A codon optimized sequence for the GLP-1 [receptor](https://2022.igem.wiki/ucsc/glossary#Receptor "Receptor") ([GLP-1 R](https://2022.igem.wiki/ucsc/glossary#GLP-1-R "GLP-1-R")) was designed for *E. coli* to test the [binding affinity](https://2022.igem.wiki/ucsc/glossary#Binding-Affinity "Binding-Affinity") of our Ex-4 in both *E. coli* and *S. cerevisiae*. This sequence was inserted into the same pET28:GFP backbone as the Ex-4 insert for *E. coli*. The gene insert for the GLP-1 R includes: BsaI restriction enzyme site, Shine-Dalgarno sequence, His-tag, linker sequence, [TEV](https://2022.igem.wiki/ucsc/glossary#TEV "TEV") cut site, and GLP-1 R codon optimized sequence. The insert includes a TEV cut site instead of an enterokinase cut site, but both have equivalent proteolytic functions. We inserted an alanine in between the TEV cut site and the GLP-1 R sequence because [TEV protease](https://2022.igem.wiki/ucsc/glossary#TEV-protease "TEV-protease") will only cut before serine, glycine, alanine, methionine, cysteine, or histidine amino acids [[7](#Citation-Accordion)]. To learn more about each of the components of the GLP-1 R gene insert, check out our [Contributions](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27contribution%27%29%20%7D%7D) page!

**Build:** The GLP-1 R was inserted into the pET28:GFP backbone through GGA. Initial assembly protocol suggested a 1:1 ratio of insert to backbone. We transformed our GGA assembled plasmids into DH10B *E. coli*, and incubated overnight with the expectation of seeing colonies the next day. To confirm the colonies we grew contained successfully ligated GGA plasmids, we performed colony PCR and gel electrophoresis. The GLP-1 R insert is 505 base pairs long, so using a 10kb ladder we were expecting bands to show up near the bottom of the gel.

**Test & Learn:** Our gel electrophoresis had bands well below the expected position, indicating that our GGA was not successful in correctly assembling the GLP-1 R plasmid. The bands traveled far enough down the gel that they seemed to be 100 base pairs long. We hypothesized that our PCR primers ligated together, creating [primer dimers](https://2022.igem.wiki/ucsc/glossary#Primer dimers "Primer dimers"), and the GLP-1 R sequence was not successfully inserted into the pET28:GFP backbone.

##### **Second Iteration**

**Design:** To test this hypothesis, we performed GGA with a 2:1 ratio of insert to backbone with the hope that increasing the number of GLP-1 R inserts would increase the probability of successful plasmid construction.

**Build & Test:** We transformed these GGA reactions into DH10B *E. coli* as we had done previously, and let them grow overnight. We performed colony PCR and gel electrophoresis to determine if the new GGA reactions were successful.

**Learn:** The gel electrophoresis showed the same bands as the previous iteration; the GGA primers formed primer dimers and the pET28:GFP backbone annealed to itself. We then decided to send our GLP-1 R insert and pET28:GFP backbone to sequencing to determine if the ends of the backbone or insert degraded, thus preventing proper plasmid assembly. The sequencing results for the backbone indicated that our backbone did not degrade, however, the sequencing results for the insert were inconclusive.

##### **Third Iteration**

**Design:** We decided to try 2:1 GGA again, but this time, use fresh reagents and amplify our GLP-1 R insert to ensure the GLP-1 R insert's ends are correct.

**Build:** We performed PCR to amplify our GLP-1 R insert and used this amplified insert in the 2:1 GGA reaction. These GGA reactions were transformed into DH10B *E. coli* and incubated overnight. We performed colony PCR and gel electrophoresis as we had done in the last iteration.

**Test:** For the first time, we saw bands at the expected position of around 500 bases! We excitedly performed miniprep on those colonies to extract the plasmid and sent them to sequencing to further confirm that the plasmid was constructed correctly. Sequencing results concluded that we properly assembled the GLP-1 R insert into the pET28:GFP backbone. These iterations taught us that GGA is finicky and can fail in multiple ways. Our success came from improving upon multiple iterations of the engineering cycle. Our next steps were then transforming into C41 *E. coli*, and performing IMAC and SDS-PAGE.

**Learn:** The SDS-PAGE had a band in the pellet at 17[kD](https://2022.igem.wiki/ucsc/glossary#kD "kD"), the expected weight of the GLP-1 R. This suggests that the receptor is insoluble in *E. coli*. Our next steps include refolding the receptor in *E. coli*, so it will bind correctly to our Ex-4.

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#### **Binding Affinity Assay**

##### **First Iteration**

**Design:** To test that our recombinant Ex-4 proteins function as expected, we planned an Octet binding affinity assay between Ex-4 and GLP-1 R. The [Octet machine](https://2022.igem.wiki/ucsc/glossary#Octet-Machine "Octet-Machine") determines the [dissociation constant](https://2022.igem.wiki/ucsc/glossary#Dissociation-Constant "Dissociation-Constant") (kd) of a protein-[ligand](https://2022.igem.wiki/ucsc/glossary#Ligand "Ligand") interaction using antibodies and a fiber optic biosensor [[8](#Citation-Accordion)]. First, this test requires the removal of one of the binding partner’s His-tag using a protease. Next, Penta-His antibodies on the biosensor tip attach to our His-tagged protein. Then, the tip submerges into the non-tagged protein sample, allowing binding reactions to occur. We decided to remove the *E. coli* Ex-4 His-tag and attach our purchased GLP-1 R on the Octet biosensor for our first round of testing.

**Build:** Contaminating salt content was removed from the chosen His-tagged Ex-4 samples using diafiltration with 3kD spin filters. After our Ex-4 was resuspended in Tris-HCL buffer, we incubated the protein with enterokinase overnight to cleave Ex-4’s His-tag. Batch purification isolated tag-free Ex-4, which was then analyzed via spectrophotometry at A280 to determine protein concentration.

**Test & Learn:** Protein concentration of our sample was around 0.001mg/mL, and did not indicate successful isolation of Ex-4. The 3kD spin filters we used during diafiltration may not have collected our 6.4kD Ex-4, as indicated by the DuBois lab who had similar issues isolating small proteins.

##### **Second Iteration**

**Design:** Since a 1kD spin filter would have been difficult for our team to obtain, we hypothesized the His-tags may be kept on both binding partners. Ensuring the Octet biosensor was fully saturated in His-tagged GLP-1 R, and the Ex-4’s His-tag was shielded using a His-tag antibody, we believed an accurate binding reaction would still occur.

**Build & Test:** We performed the binding affinity assay using an Octet machine under the conditions described on the previous page. Controls for this assay included an unloaded sensor submerged into Ex-4 and a GLP-1 R loaded sensor submerged into His antibodies.

**Learn:** The graph produced in real time from this assay indicated this experiment was not successful, suggesting binding did not occur. Our next step is to obtain a 1kD spin filter to properly capture Ex-4 so we can perform the binding affinity assay under the proper condition where only one binding partner has a His-tag.

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#### **Modeling**

**Design:** The first model we wrote predicted Ex-4's binding affinity in the presence of its endogenous competitor, [GLP-1](https://2022.igem.wiki/ucsc/glossary#GLP-1 "GLP-1"), aptly named the competitive binding model. A crucial step in this process was deciding which formula best described our system, where both Ex-4 and GLP-1 generate the desired pharmacological effect. After speaking with Dr. Michael Stone, a biochemistry professor at UC Santa Cruz, we chose a variation of the Cheng-Prusoff equation [[7](#Citation-Accordion)] for its history of representing competitive binding interactions. For all parameters involved (e.g. IC50, Ki), we found literature values by searching through online databases such as PubMed and Google Scholar.

**Build and Test:** We programmed the competitive binding model in Python 3 using Jupyter Notebook. While we initially attempted to use MATLAB, Python was ultimately the better choice due to our team's past experience with the language. We ran the code and adjusted our graph’s specifications for clarity of our model.

**Learn:** We learned this model does not appropriately represent our system because it does not account for the effect of GLP-1 and Ex-4 both producing insulin. The Cheng-Prusoff equation tells us the concentration at which Ex-4 inhibits 50% of the natural ligand binding. We changed our equation to [fractional occupancy](https://2022.igem.wiki/ucsc/glossary#Fractional-Occupancy "Fractional-Occupancy") because it tells us how many ligands are bound compared to the total amount of receptors in a system. This way, we would be able to more accurately compare the competitive binding between Ex-4 and GLP-1.

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## Citations

1. [1] D. L. Shis and M. R. Bennett, “Library of synthetic transcriptional AND gates built with split T7 RNA polymerase mutants,” *Proceedings of the National Academy of Sciences*, vol. 110, no. 13, pp. 5028–5033, Mar. 2013, doi: [10.1073/pnas.1220157110](https://www.pnas.org/doi/full/10.1073/pnas.1220157110).
2. [2] C. Engler, R. Gruetzner, R. Kandzia, and S. Marillonnet, “Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes,” *PLOS ONE*, vol. 4, no. 5, p. e5553, May 2009, doi: [10.1371/journal.pone.00055533](https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0005553).
3. [3] M. E. Lee, W. C. DeLoache, B. Cervantes, and J. E. Dueber, “A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly,” *ACS Synth. Biol*., vol. 4, no. 9, pp. 975–986, Sep. 2015, doi: : [10.1021/sb500366v](https://pubs.acs.org/doi/10.1021/sb500366v).
4. [4] “Frontiers | Golden Gate Cloning-Compatible DNA Replicon/2A-Mediated Polycistronic Vectors for Plants.” [https://www.frontiersin.org/articles/10.3389/fpls.2020.559365/full](https://www.frontiersin.org/articles/10.3389/fpls.2020.559365/full%20) (accessed Oct. 05, 2022).
5. [5] “Codon Optimization Tool | IDT,” *Integrated DNA Technologies.* <https://www.idtdna.com/pages/tools/codon-optimization-tool>.
6. [6] S. J. Jung *et al.*, “In-vivo half-life and hypoglycemic bioactivity of a fusion protein of exenatide and elastin-based polypeptide from recombinant Saccharomyces cerevisiae,” *Journal of Biotechnology*, vol. 303, pp. 16–24, Sep. 2019, doi:  [10.1016/j.jbiotec.2019.06.304](https://doi.org/10.1016/j.jbiotec.2019.06.304).
7. [7] Y. Cheng and W. H. Prusoff, “Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction,” *Biochem Pharmacol*, vol. 22, no. 23, pp. 3099–3108, Dec. 1973, doi: 10.1016/0006-2952(73)90196-2 [10.1016/0006-2952(73)90196-2](https://doi.org/10.1016/0006-2952%2873%2990196-2).
8. [8] “Octet® HIS1K Biosensors,” Sartorius. [https://www.sartorius.com/en
   /products/protein-analysis/octet-bli-detection/biosensors-chips-kits/antipenta-his-his1k-biosensors](https://www.sartorius.com/en/products/protein-analysis/octet-bli-detection/biosensors-chips-kits/antipenta-his-his1k-biosensors)  Accessed: Aug. 28, 2022.

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