iGEM 2024 — Team virginia

# **Notebook**

[Click to read content](#preparation)

![](https://static.igem.wiki/teams/5178/headers/barry/barry-notebook.webp)

* [Preparation](#preparation)
* [June](#june)
* [July](#july)
* [August](#august)
* [September](#september)
* [October](#october)

## Preparation

## 11/27/23 - 5/31/24

**November 27, 2023:**

* Applications for iGEM 2024 due

**December 3-8, 2023:**

* Interviews for iGEM

**December 13, 2023:**

* iGEM acceptances sent

**January 18, 2024:**

* First time meeting everyone!
* First day of Synthetic Biology class (BIOL 4770)

**April 11, 2024:**

* Group chat formed!

**April 26, 2024:**

* Team voted on leadership positions

- **April 29, 2024:**
  * First official team meeting online
  * Discussed summer schedule**May 8, 2024:**
  * Met online to discuss project ideas
  * Read through proposals from class**May 10, 2024:**
  * Established OneDrive**May 15, 2024:**
  * Discussed feedback on existing project proposals**May 24, 2024:**
  * Brainstormed new project ideas**May 30, 2024:**
  * First in-person meeting in lab**May 31, 2024:**
  * Finalized project idea

![A preliminary whiteboard diagram of our project. The left reads: 'PEI causes no lipase. PEI + Pancreatic cancer.' A vertical flowchart is in the middle and the first item is: 'Treatment = Pancrelipase (from pigs, bad).' An arrow points from here to the next item: 'lipase from yeast which makes amylase and protease (p. Pastoris). And lipase from e. coli (doesn't need glycosylation).' A bracket surrounding these says: 'Bad/not good for pancreatitis, low levels of lipase.' The next arrow points to: 'human pancreatic lipase from B. subtilis (common probiotic and good for pancreatitis). Makes amylase and protease.' The last arrow points to: 'Plus switch? Lac operon (ex)?'.](https://static.igem.wiki/teams/5178/notebook/img-6974.jpeg)

##### *Brainstorming our project*

## June

## Week 1 (6/3/24-6/7/24)

**June 3:**

* Decided on our project topic
* Toured our lab spaces for the summer

**June 4-June 7:**

* Started biobricking our constructs
* Figured out glucose switch and read literature on transformation of human pancreatic lipase into bacteria
* First lab meeting on June 6

## Week 2 (6/10/24-6/14/24)

**June 10-12:**

* Determined assembly method

+ Considered all options, including Standard, Golden Gate, and 3A
+ Settled on Gibson + Standard Asssembly for our final design
+ Finalized restriction enzymes to be used

* Worked on kill-switch mechanism

**June 13:**

* Finalized parts to order
* Contacted experts in ethics and experts in pancreatic conditions
* Started working on logo and visual ideas for the project and wiki

**June 14:**

* Decided project/team name
* Developed project logo
* Worked on project description for wiki
* Ordered parts

## Week 3 (6/17/24-6/21/24)

**June 17:**

* Started working on handbook and DIY synthetic biology kit
* Started putting content on wiki
* Prepared transformation buffer
* Started pre-culture of *E. coli* DH5α to prepare for competent cell protocol
* Started developing models using values from literature

**June 18:**

* Ordered primers
* Completed a majority of handbook
* Developed a growth curve for *E. coli* DH5α using a spectrophotometer
* Inoculated 500 mL flask using pre-culture

**June 19:**

* Attempted to measure growth curve for 500 mL flask

+ Discovered an error with the culture

* Made a new pre-culture with *E. coli* JM109

**June 20:**

* Inoculated two 500 mL flasks with *E. coli*J M109 pre-culture
* Developed "What is Synthetic Biology" video
* Started writing questions for interviews

**June 21:**

* Performed competent cell protocol using *E. coli* JM109 culture

+ Test for competency was unsuccessful due to faulty plasmid

* Practiced PCR and gel electrophoresis using spare materials
* Ran mock gel electrophoresis experiment for synthetic biology handbook

## Week 4 (6/24/24-6/28/24)

**June 24:**

* Attempted second transformation to test competent cells using pUC19
* Completed interview with Mrs. Eleanor Nicholson, an expert in homeschool education
* Added teacher's guide to synthetic biology handbook
* Gained introduction into neural networking topics for modeling
* Updated preliminary model on lipase and colipase using values from literature

**June 25:**

* Analyzed plate of transformed cells from June 24

+ Transformation was successful!

* Completed interview with Dr. Elizabeth Barnes, expert in healthcare philosophy
* Continued developing teacher's guide
* Incorporated glucose concentrations into model kinetics

**June 26:**

* Counted transformed cells using ImageJ

+ Calculated transformation efficiency

* Consolidated protocols, including PCR, gel electrophoresis, Western blot, etc
* Continued to work on different interactions fo the glycolysis and lipase expression pathways in model

+ Introduced new species (ex: T7RNAP transcription, GlcT RAT binding) and new rates

* Filmed applesauce enzyme lab for synthetic biology handbook

**June 27:**

* Added repression mechanism for LacI and T7RNAP genes to model
* Completed interview with Dr. Ray Wadlow, expert in pancreatic cancer
* Updated synthetic biology handbook based on peer feedback

**June 28:**

* Started investigating mechanism of LacI repression of T7RNAP in model
* Started working on IRB forms for synthetic biology handbook focus group
* Developed experimental plan for upcoming week

## July

## Week 5 (7/1/24-7/3/24)

**July 1:**

* Hydrated primers
* Plated bacterial stab with pBS1C primer
* Considered sensitivity of lacI in model
* Started CITI training in behavioral sciences research for IRB approval
* Completed interview with Dr. Deborah Froh, an expert in pediatric cystic fibrosis, Dr. Kevin Lonabaugh, a clinical pharmacist, Kasey Wilburn, a registered dietitian, and Terri Quinan, executive director of the Virginia chapter of the Cystic Fibrosis Foundation.

**July 2:**

* Submitted first draft of IRB protocol
* Developed infographic explaining EPI for Instagram
* Restreaked plates with pBS1C plasmid
* Hydrated and diluted parts to create working stock for Gibson assembly

**July 3:**

* Received feedback on IRB protocol; submitted second draft
* Analyzed plates streaked yesterday and found that there were no single colonies

## Week 6 (7/8/24-7/14/24)

**July 8:**

* Started making captions for videos
* Added alt-text to wiki pictures to increase accessibility for screen-reader usage
* Investigated use of alphafold for 3D models integrated into computational models
* Simplified MatLab SymBiology model
* Started training large language model to simplify iGEM registry navigation
* Streaked chloramphenicol and ampicillin plates with pBS1c
* Ordered plasmid pHY300PLK for kill-switch

**July 9:**

* Performed PCR and purification on parts 3B and 5A twice

+ Both attempts were unsuccessful, but taught the team valuable skills

* Investigating how phosphorylation changes protein shape in model
* Began coding database scraper for key components of iGEM registry for easier filtering
* Modified general wiki design for increased accessibility

**July 10:**

* Aimed to determine ideal concentration of chloramphenicol to add to LB-ampicillin media
* Spoke with a panel of nine oncology dietitians
* Completed interview with Dr. Shahid Ali, an expert in gastroenterology
* Consolidated computational models

**July 11:**

* First mini-prep of pBS1C
* Observed overnight pBS1C liquid cultures from July 10

+ Determined no growth of cells at any concentration of chloramphenicol
+ Investigated plasmid antibiotic resistance variability based on chassis
+ Streaked pBS1C onto ampicillin plates

* Inoculated LB-chloramphenicol media with pACYC (chloramphenicol-resistant plasmid)

+ Concentrations of chloramphenicol: 12.5 µg/mL, 25 µg/mL, 50 µg/mL, 75 µg/mL, 100, µg/mL

* Poured new chloramphenicol plates at concentrations of 5 µg/mL, 25 µg/mL, 35 µg/mL
* Completed interview with Dr. Daniel Strand, an expert in pancreatic cancer

**July 12:**

* Observed ampicillin plates with pBS1C
* Streaked onto chloramphenicol plates made July 11
* Linearized mini-prepped pBS1C from July 11

+ Ran linearized vector on gel to determine length (~7kb)

**July 14:**

* Inoculated overnight cultures of pBS1C

+ Three cultures of 100 µg/mL ampicillin in 2 mL LB
+ Three cultures of 50 µg/mL ampicillin in 2 mL LB

## Week 7 (7/15/24-7/19/24)

**July 15:**

* Mini-prepped one tube of pBS1C overnight liquid culture

+ Low yield due to expired materials and lack of experience

* Restriction digested with EcoRI-HF and PstI-HF in CutSmart Buffer
* Made glycerol stocks with the remainder of the overnight culture
* Inoculated new overnight liquid cultures: three cultures of 100 µg/mL ampicillin in 2 mL LB

**July 16:**

* Mini-prepped overnight culture from July 15

+ Yield from mini-prep was higher than previous attempt but still below desired range

* Restriction digested with EcoRI and PstI-HF in NEB r3.1 Buffer

+ Set up two reactions of 50 µL each

* Ran gel with two 20 µL wells from one digested reaction
* Gel was melted and unstable, and bands were smeared
* Poured a new gel to run the following day
* Inoculated new overnight liquid cultures of pBS1C: four cultures of 100 µg/mL ampicillin in 2 mL LB
* Hydrated lyophilized pHY300PLK in nuclease-free water and transformed into competent *E. coli* JM109

**July 17:**

* Reran gel from July 16 with mini-prepped and digested template DNA

+ Image of gel using Gel Doc EZ Imager appeared to have bright bands
+ After performing gel extraction, yield was very low

* Reattempted mini-prep to perfect technique and produce higher yield

+ Yield was higher than previous attempts but still below desired range

* Restriction digested mini-prep with EcoRI-HF and PstI-HF in CutSmart Buffer

+ Calculations for restriction digest were incorrect and thus only 300 ng (instead of 1000 ng) of DNA were used
+ Performed another restriction digest with correct calculation (1000 ng DNA)

* Ran gel with wrongly digested template DNA

+ Image of gel using Gel Doc EZ Imager appeared to have bright bands
+ Will cut out bands and perform gel extraction on July 18

* Hydrated lyophilized *B. subtilis* 168 using potato media and plated onto potato-agar plates
* Inoculated three overnight liquid cultures of pHY300PLK in 100 µg/mL ampicillin in 2 mL LB

**July 18:**

* Ran gel with correctly digested template DNA from July 17
* Gel extraction performed on both correctly digested template and wrongly digested template from July 17

+ Yields for three out of four extractions were low, but one was high enough for Gibson assembly

* Attempted Gibson assembly with positive control and "A" parts for "Gibson plasmid A"
* Transformed competent *E. coli* JM109 with "Gibson plasmid A" and positive control, and incubated
* Observed *B. subtilis* 168 plates from July 17

+ Restreaked plates, seeking single colonies

* Inoculated new overnight liquid cultures of pBS1C

+ 1. Two cultures containing 100 µg/mL ampicillin in 4 mL LB were grown for seven hours, and then 170 µg/mL chloramphenicol was added
+ 2. Two cultures containing 100 µg/mL ampicillin and µg/mL chloramphenicol in 4 mL LB were grown overnight
+ 3. Two cultures containing 100 µg/mL ampicillin in 4 mL LB were grown overnight

**July 19:**

* Attempted mini-prep of cultures 3 from July 18 using new Zymo mini-prep kits

+ Yield was very high compared to previous attempts

* Restriction digested new mini-prepped pBS1C using EcoRI-HF and PstI-HF in CutSmart Buffer
* Learned gel extraction techniques from Ms. Parker
* Attempted gel extraction on restriction digested pBS1C from this morning

+ Yield was higher than previous attempts

* Presented at UVA Bridge Program to educate incoming engineering students about iGEM

**July 21:**

* Inoculated LB-ampicillin media with pHY300PLK and pBS1C

## Week 8 (7/22/24-7/26/24)

**July 22:**

* Attempted Gibson assembly with “A” parts for “Gibson plasmid A” and “B” parts for “Gibson plasmid B” using gel extracted vector from 7/19
* Transformed competent *E. coli* JM109 with ”Gibson plasmid A” and “Gibson plasmid B”
* Mini-prepped pBS1C and pHY300PLK

**July 23:**

* Restriction digested mini-prepped pBS1C from 7/22 with EcoRI-HF and PstI-HF in NEB rCutSmart Buffer

+ Ran digested products through a gel

* Hydrated parts 3A and GFP
* Inoculated overnight liquid cultures of “Gibson plasmid A” and “Gibson plasmid B”
* Inoculated overnight liquid culture of *B. subtilis* 168
* Transformed *E. coli* JM109 with “Gibson plasmid A” again

**July 24:**

* Performed analytical digest of “Gibson plasmid A” and “Gibson plasmid B” to verify sequence

+ Performed gel extraction of P43-NC06-MazE-RBS-MazF-B0015

* Restriction digested Pxyl-RBS-MazE-B0015 and P43-RBS-XylR-B0015
* Restriction digested “Gibson plasmid A1”, “Gibson plasmid B2”, and “Gibson plasmid B3”
* Restriction digested and performed gel extraction on pBS1C

**July 26:**

* Reattempted Gibson assembly for “Gibson plasmid A” and “Gibson Plasmid B” using gel extracted pBS1C from 7/25
* Transformed competent *E. coli* JM109 with "Gibson plasmid A" and “Gibson plasmid B” using two different protocols:

+ NEBuilder HiFi DNA Assembly Transformation Protocol – O
+ High Efficiency Transformation Protocol for C269871 – E

* Restriction digested pHY300PLK with HindIII and BamHI

+ Ran on a gel with Pxyl-RBS-MazE-B0015 and P43-RBS-XylR-B0015
+ Performed gel extraction on all parts and plasmid

* Mini-prepped “Gibson plasmid A2 colony 2” from liquid culture inoculated on 7/24
* Restriction digested “Gibson plasmid A2 colony 2” with EcoRI-HF and PstI-HF

## Week 9 (7/29/24-8/2/24)

**July 29:**

* Restriction digested pBS1C with NotI instead of PstI for Gibson assembly, and performed gel extraction
* Attempted Gibson assembly for “Gibson plasmid B”
* Transformed competent *E. coli* JM109 with "Gibson plasmid B”
* Updated synthetic biology handbook using feedback from homeschool expert Mrs. Sudhita Kasturi
* Contacted homeschool groups to distribute survey

**July 30:**

* Restriction digested pBS1C with NotI instead of PstI for Gibson assembly, and performed gel extraction
* Attempted Gibson assembly for “Gibson plasmid A”
* Transformed competent *E. coli* JM109 with "Gibson plasmid A”
* Inoculated overnight liquid cultures of “Gibson plasmid B”

**July 31:**

* Mini-prepped “Gibson plasmid B”, restriction digested, and ran through a gel to verify
* Poured xylose plates for kill-switch testing

**August 1:**

* Mini-prepped “Gibson plasmid A”, restriction digested, and ran through a gel to verify
* Attempted ligation of parts Pxyl-RBS-MazE-B0015, P43-RBS-XylR-B0015, and pHY300PLK
* Performed PCR on parts 4A (database cre-sites) and 4B (paper cre-sites) with primers containing Gibson overhangs

**August 2:**

* Attempted standard assembly of 1A into pHY300PLK
* Sent samples of “Gibson plasmid A” and “Gibson plasmid B” for DNA sequencing
* Restriction digested 4A (database cre-site) and 4B (paper cre-sites)
* Attempted Gibson assembly with 4A (database cre-sites) and 4B (paper cre-sites)
* Transformed competent *E. coli* JM109 with product

## August

## Week 10 (8/5/24-8/9/24)

**August 5:**

* Restriction digested pHY300PLK and Part 1A with EcoRI-HF and AgeI-HF
* Performed gel extraction on pHY300PLK and Part 1A
* Attempted ligation and transformation into competent *E. coli* JM109
* Performed PCR on GFP and ran through gel
* Inoculated overnight liquid cultures of pHY300PLK-Pxyl-RBS-MazE-B0015-P43-RBS-XylR-B0015 and "Gibson plasmid A1"

**August 6:**

* Mini-prepped “Gibson plasmid A1”, restriction digested mini-prep with EcoRI-HF and AgeI-HF, and ran through a gel
* Performed gel extraction of Part 1A
* Mini-prepped pHY300PLK, restriction digested with NdeI to verify, and ran through a gel
* Performed gel extraction of P43-RBS-XylR-B0015
* Performed inverse PCR on “Gibson plasmid A1”

**August 8:**

* Mini-prepped “Gibson plasmid B2.1”
* Attempted ligation of Part 3A into mini-prep of Gibson B2.1 colony and transformed into competent *E. coli* JM109
* Attempted ligation of Part 1B, Part 2B, and Part 3B, and Gibson plasmid A3.2 and transformed into competent *E. coli* JM109
* Mini-prepped pHY300PLK-1A, linearized with EcoRI-HF, and ran through a gel to verify construct, but it was unsuccessful

## Week 11 (8/11/24-8/16/24)

**August 10:**

* Received sequencing for constructs

+ Gibson Plasmid B2.1 contains 1B, 2B, 3B, 4B – pBS1C-1B-2B-3B-4B
+ Gibson Plasmid B2.2 contains 2B, 3B, 4B – pBS1C-2B-3B-4B

**August 11:**

* Inoculated overnight liquid culture of pBS1C-1A, pBS1C-1B-2B-3B-4B, and pBS1C-2B-3B-4B

**August 12:**

* Mini-prepped overnight liquid cultures from 8/11
* Performed inverse PCR and purification on backbone for Gibson assembly

+ Ran on gel to verify

* Performed PCR and purification on Part 3A
* Restriction digested pBS1C-1B-2B-3B-4B and 3A with PstI-HF and RsrII-HF

+ Performed gel extraction of backbone and part to attempt ligation

* Restriction digested pBS1C-2B-3B-4B mini-prep and Part 3A with BspDI and PstI-HF

+ Performed gel extraction of backbone and part to attempt ligation

* Restriction digested pBS1C-1B-2B-3B-4B and 3A with BspDI and PstI-HF

+ Performed gel extraction of backbone and part to attempt ligation

**August 13:**

* Performed inverse PCR and purification on backbone
* Attempted ligation of pBS1C-1B-2B-3B-4B with 3A to create pBS1C-1B-3A

+ Transformation into *E. coli* JM109

* Attempted ligation of pBS1C-2B-3B-4B with 3A to create pBS1C-2B-3A

+ Transformation into *E. coli* JM109

* Attempted ligation of pBS1C-1B-2B-3B-4B and 3A to create pBS1C-1B-2B-3A

+ Transformation into *E. coli* JM109

* Attempted Gibson assembly of pBS1C-1A with part 2A, 3A, and 4A

+ Transformation into *E. coli* JM109

**August 14:**

* Poured phenol red lipase plates
* Inoculated overnight cultures of Gibson A attempt (pBS1C-1A-2A-3A-4A)
* Observed no growth on ligation plates

**August 15:**

* Prepare samples of Gibson A attempt to send for DNA sequencing
* Made glycerol stocks of all transformations of Gibson A Attempts
* Restriction digested pBS1C-1B-2B-3B-4B and 3A with PstI-HF and RsrII-HF

+ Performed agarose ligation

* Restriction digested pBS1C-2B-3B-4B and 3A with BspDI and PstI-HF

+ Performed agarose ligation

* Restriction digested pBS1C-1B-2B-3B-4B and 3A with BspDI and PstI-HF

+ Performed agarose ligation

**August 16:**

* Restriction digested Gibson A attempt colonies 1-6 with KpnI-HF and ran on a gel to verify
* Sent samples for DNA sequencing

## Week 12 (8/22/24-8/2/23)

**August 22:**

* Digested pBS1C with AvrII and BBvCI
* Ran on gel
* Performed agarose ligation (3A + pBS1C-1B-2B-3B-4B)and transformed into *E. coli* JM109

+ No colonies grew

## Week 13 (8/26/24-9/1/24)

**August 26:**

* Mini-prepped pBS1C for digestion

**August 27:**

* Inoculated overnight liquid cultures of pBS1C-1B-2B-3B-4B
* Troubleshooted ligation by testing with ligase, water, and vector

**August 28:**

* Mini-prepped pBS1C-1B-2B-3B-4B, and restriction digested it and 3A with BspDI and PstI, and ran on gel
* Performed PCR and purification on Part 3A
* Transformed ligation tests from 8/27 into competent *E. coli*JM109
* Restriction digested pBS1C with AvrII and BBvCI and performed gel ligation with 4B

**August 29:**

* Performed PCR and purification on 3A and ran on gel to verify
* Attempted ligation of pBS1C-4A-GFP (database cre-sites) and pBS1C-4A-GFP (paper cre-sites)

**August 31:**

* Inoculated overnight liquid cultures of pBS1C-4A-GFP (database cre-sites)

**September 1:**

* Mini-prepped pBS1C-4A-GFP (database cre-sites), restriction digested with PstI, and ran on gel

## September

## Week 14 (9/2/24-9/8/24)

**September 2:**

* Performed PCR of pBS1C-4A (database cre-sites), pBS1C-4B (paper cre-sites), and GFP with Q5 high-fidelity DNA polymerase

+ Ran gel of PCR products, did not match expected results

* Performed PCR of pBS1C-4A(database cre-sites), pBS1C- 4B (paper cre-sites), and GFP with Phusion high-fidelity DNA polymerase

+ Ran gel of PCR products, gel looked as expected

**September 3:**

* Gel extracted PCR product that was performed with Phusion high-fidelity DNA from 9/2
* Inoculated overnight liquid cultures of pHY300PLK for ligase troubleshooting
* Attempted Gibson assembly of pBS1C- 4B-GFP (database cre-sites) and P434A-B0015 (paper cre-sites) and transformed into *E. coli* JM109

+ Only a few colonies grew

**September 4:**

* Attempted Gibson assembly again with pBS1C- P43-4A-GFP (database cre-sites) and 4B (paper cre-sites) using a different Gibson premix and transformed into *E. coli*JM109

+ Only a few colonies grew

* Mini-prepped pHY300PLK

**September 5:**

* Performed PCR of pBS1C-4A (database cre-sites), pBS1C-4B (paper cre-sites) with double the reaction volume

**September 6:**

* Ran PCR products from 9/5 on gel and gel extracted
* Attempted Gibson assembly of pBS1C-P4A-GFP (database cre-sites), pBS1C-4B-GFP (paper cre-sites) and transformed into *E. coli* JM109

+ Only a few colonies grew

* Restriction digested pBS1C-1B-2B-3B-4B and pHY300PLK with EcoRI and AgeI to obtain part 1B for pHY300PLK- 1B ligation, and ran through gel
* Performed gel extraction for vector and insert

+ Used these with new ligation kit, the NEB Quick Ligation Kit, to ligate pHY300PLK-1A and transformed into competent *E. coli* JM109

**September 7:**

* Performed PCR of GFP with double the reaction volume
* Attempted Gibson assembly of pBS1C-4A-B0015-GFP (database cre-sites), pBS1C-4B-GFP (paper cre-sites) with positive control and transformed into *E. coli* JM109

+ Positive control had over 20 colonies, but the experimental plate had less than 10

* Attempted Gibson assembly of pBS1C-4A-GFP (database cre-sites), pBS1C-4B-GFP (paper cre-sites) with positive control and transformed into *E. coli* JM109

## Week 15 (9/9/24-9/15/24)

**September 9:**

* Inoculated overnight liquid cultures of pHY300PLK-1A

**September 10:**

* Mini-prepped pHY300PLK-1A
* Restriction digested pHY300PLK-1A with PstI-HF and ran on gel to verify

**September 11:**

* Inoculated overnight liquid cultures of 6 pBS1C-P4A-GFP (database cre-sites) colonies and 6 pBS1C-4B-GFP (paper cre-sites) colonies obtained through Gibson assembly and transformation

**September 12:**

* Mini-prepped pBS1C-4A-GFP (database cre-sites) and pBS1C-4B-GFP (paper cre-sites) from previous day's overnights
* Restriction digested each mini-prep with BamHI-HF and ran on gel

**September 13:**

* Restriction digested pBS1C with AvrII and BbvCI and ran on gel to obtain vector
* Performed gel extraction of vectors

**September 14:**

* Attempted Gibson assembly of pBS1C- 4A-GFP (database cre-sites) and pBS1C- 4B-GFP (paper cre-sites) using backbone from 9/13

+ Transformed into *E. coli* JM109 using electroporation
+ 20+ colonies grew for each assembly

## Week 16 (9/16/24-9/21//24)

**September 16:**

* Mini-prepped 2 colonies each of pBS1C- 4A-GFP (database cre-sites) and pBS1C- 4B -GFP (paper cre-sites) from 9/14
* Restriction digested mini-prepped samples with BamHI-HF to verify GFP insert and ran on gel

+ Gel looked as expected

**September 17:**

* Repeat restriction digest and verification from 9/16, gel again looks as expected

**September 20:**

* Troubleshooting ligation using pKK1095 and pKK1395

+ Restriction digested using SacI and SalI
+ Ran on gel
+ Attempted ligation with T4 DNA ligase, Hi-T4 DNA ligase, and NEB Quick Ligase at room temperature, incubated temperatures, with different ligation periods, and with and without heat inactivation of ligase

* Streaked *B. subtilis* 168 onto Terrific Broth agar plate
* Transformed ligation troubleshooting plasmids into *E. coli* JM109

**September 21:**

* Transformed pBS1C-4A-GFP (database cre-sites) and pBS1C-4B-GFP (paper cre-sites) into *B. subtilis* 168 and plated onto ampicillin plates.

## Week 17 (9/23/24-9/29/24))

**September 27:**

* Rehydrated Gibson primers for (Pxyl-MazE), (P43-NC06-MazE-MazF), and (P43-XylR) to 100 µm and made working stocks of 10 µm concentration
* Rehydrated new Pxyl-RBS-MazE-B0015, P43-NC06-MazE-RBS-MazF-B0015, and P43-RBS-XylR-B0015 to 100 ng/µL
* Performed PCR of Pxyl-RBS-MazE-B0015, P43-NC06-MazE-RBS-MazF-B0015, and P43-RBS-XylR-B0015 with Gibson primers

+ Performed PCR purification
+ Ran analytical gel

* Made xylose-amp plates

**September 29:**

* Restriction digested pHY300PLK with EcoRI and HindIII-HF
* Ran gel and gel extracted vector for Gibson assembly

## October

## Week 18 (10/1-10/8)

**October 1:**

* Restriction digested plasmid extracted from *B. subtilis* 168 with EcoRI and ran on gel

**October 4:**

* Attempted Gibson assembly of pHY300PLK-5A-5B-5C
* Transformed pHY300PLK-5A-5B-5C into *E. coli* DH5α and *E. coli* JM109

+ Plated onto 1% and 2% xylose plates

* Performed PCR with GFP primers and plasmids extracted on 10/1, ran gel

+ Inoculated overnight cultures in *E. coli* JM109 in LB amp

**October 5:**

* Washed pellets from overnight started on 10/4, inoculated new overnight cultures in CSE minimal media

**October 6:**

* Inoculated overnight cultures of pHY300PLK-5A-5B-5C

**October 7:**

* Spun down cells from 10/5, screened for fluorescence
* Mini-prepped 11 colonies from pHY300PLK-5A-5B-5C from 10/6

+ Restriction digested with SexAI
+ Ran on gel and imaged

* Made chloramphenicol stocks and plates
* Hydrated primers for parts 1C and 2C
* Performed gradient PCR for parts 1C and 2C to determine optimal annealing temperatures

**October 8:**

* Performed PCR of 1C and 2C
* Restriction digested pBS1C
* Ran 1C, 2C, and pBS1C on gel
* Performed gel extraction of 1C, 2C, and pBS1C
* Attempted Gibson assembly of pBS1C-1C-2C and transformed into *E. coli* DH5α

## Week 19 (10/9-10/11)

**October 9:**

* Transformed *B. subtilis* 168 with pBS1C-4A-GFP and pBS1C-4B-GFP, pBS1C-1C-2C, and pBS1C
* Inoculated overnight cultures of pBS1C-1C-2C in *E. coli DH5α*

**October 10:**

* Mini-prepped 10 overnight cultures of pBS1C-1C-2C and ran all samples on gel
* Observed no growth of pBS1C-4A-GFP or pBS1C-4B-GFP on LB-chl plates, but LB-amp plates were overgrown

**October 11:**

* Performed enzymatic lipase assay with pBS1C-1C-2C in *E. coli* DH5α

## Week 20 (10/16-10/21)

**October 16:**

* Streaked all pBS1C-1C-2C samples on plates
* Made chloramphenicol plates
* Transformed all pBS1C-1C-2C samples in *E. coli* BL21

+ Inoculated overnight cultures of pBS1C-1C-2C samples

**October 17:**

* Spun down 11/16 overnights of pBS1C-1C-2C and resuspended in SDS running buffer
* Transformed *B. subtilis* 168 with pBS1C-4A-GFP, pBS1C-4B-GFP, and pBS1C-1C-2C onto 5µg/mL LB-chl plates

**October 18:**

* Replated *B. subtilis* 168 with pBS1C-4A-GFP, pBS1C-4B-GFP, and pBS1C-1C-2C
gase at room temperature, incubated temperatures, with different ligation periods, and with and without heat inactivation of ligase * Inoculated overnight colonies with *B. subtilis* 168 colonies that grew on LB-chl

**October 19:**

* Spun down pBS1C-4A-GFP and pBS1C-4B-GFP cells from overnight started 10/18
* Made new CSE minimal media
* Grew pellets in glucose-free and glucose-rich minimal media

**October 20:**

* Screened cells from 10/19 grown in glucose-free and glucose-rich media for GFP fluorescence

+ Cells in glucose-free media show stronger fluorescence, as expected

* Performed SDS-PAGE with samples 6, 9, and 10 of pBS1C-1C-2C in *E. coli* BL21 and sample 6 of pBS1C-1C-2C in *B. subtilis* 168

**October 21:**

* Imaged SDS-PAGE gel

+ Lipase appears to be expressed in *B. subtilis* 168 sample 6 and *E. coli* BL21 samples 9 and 10!

* Based on sequencing data, it appears that procolipase is also expressed

{% endblock %}

# **Engineering**

[Click to read content](#Design)

![](https://static.igem.wiki/teams/5178/headers/barry/barry-engineering.webp)

* [Design](#Design)
* [Part Justifications](#parts)
* [Testing Plan](#test-plan)
* [Build](#build)
* [Redesign](#redesign)
* [Test](#test)
* [Safety Parts and and Engineering](#safety)

## Design

### Define the Problem

The engineering design cycle begins with the identification of a prominent problem which our team aims to address. After many days of brainstorming and research, our team discovered a lack of available and inclusive treatments for Exocrine Pancreatic Insufficiency (EPI). EPI is a condition characterized by a deficiency in digestive enzymes including pancreatic lipase, colipase, amylase, and protease. Currently, all prescribed treatments for this condition are sourced from porcine pancreases and thus are associated with various ethical concerns.

### Perform Background Research

After identifying our problem, our team aimed to learn more about the condition and current treatments through background research. We learned that EPI is a complication associated with various other conditions, including cystic fibrosis, chronic pancreatitis, type 1 diabetes, and other pancreatic conditions. We also learned more about existing pancreatic enzyme replacement therapies, commonly known as PERTs. We learned that these enzymes are derived from porcine pancreases and are thus not accessible for patients with religious or dietary restrictions. Additionally, we learned that these existing medications are not very effective and are often burdensome on patients, who may have to take up to 30 capsules daily. Our team learned more about our identified problem through online research, as well as discussions with experts in the field (doctors, synthetic biologists, dietitians, etc.).

### Identify Solution Criteria

In order to brainstorm and develop a solution, we first needed to identify constraints and specific criteria we wanted our solution to incorporate. Primarily, the key focus of our project was to eliminate the need for animals, such as pigs or cattle, as a source for pancreatic lipase and digestive enzymes. Additionally, we aim for our solution to be more effective than existing treatments, as there is a high financial and pill burden associated with current medications.

### Solution Ideation

After identifying what we wanted our ideal solution to look like, we began brainstorming and developing our solution plan. Our team discussed many rough plans and variations for our final design. Our team discussed pros and cons of using constitutive versus inducible promoters/switches, one- versus two-plasmid systems, and different tags and signal peptides for our proteins of interest.

After brainstorming various solution ideas, our team decided on a yogurt formulation containing the *B. subtilis* probiotic, which would be modified to secrete the desired digestive enzymes. This alleviates the concerns associated with current treatments, as our solution synthetically produces these enzymes, preventing the need for a treatment sourced from animals.

### Initial Design

Our novel alternative treatment for EPI planned to utilize the human pancreatic lipase gene (PNLIP) and the procolipase gene (CLPS) to synthetically produce and secrete these enzymes from their chassis, *B. subtilis*. *B. subtilis* was chosen as our chassis as it is naturally present in the gut microbiome and is thus non-immunogenic. Additionally, *B. subtilis* has been found to alleviate pancreatitis-associated complications and has not been found to exacerbate these conditions, unlike other probiotics. We planned to insert the PNLIP and CLPS coding sequences, along with specific promoters and *B. subtilis*-optimized terminators into our plasmid backbone, pBS1C. As the current treatment is consumed with each meal, we sought our design to be inducible and thus express these enzymes in response to consumption of food. Therefore, our initial circuit design utilized a complex glucose-inducible switch mechanism to induce the expression of PNLIP and CLPS. The glucose-switch mechanism is composed of two key components: a terminator/anti-terminator mechanism and a cre-promoter.

![first-circuit-design](https://static.igem.wiki/teams/5178/circuit-diagrams/1st-circuit.png)

**Figure 1.** Diagram depicting a brief overview of our circuit design. Without glucose present in the system, PNLIP and CLPS are repressed. When glucose is present in the system, PNLIP and CLPS are expressed.

In the presence of glucose, dephosphorylated GlcT binds to the RNA anti-terminator (RAT) sequence to physically hide the termination sequence, allowing for T7 RNA polymerase mRNA translation. As the promoters preceding the PNLIP and CLPS coding sequences are induced by T7 RNA polymerase, translation of this coding sequence is crucial for expression of lipase and procolipase. Simultaneously, HPrSerP-CcpA complexes form following glucose uptake in *B. subtilis* (as a result of the carbon catabolite repression pathway). These complexes bind to catabolite responsive elements (or cre sites) and repress the transcription of the lacI gene. This silences LacI production, thus lifting repression on T7 RNA polymerase, PNLIP, and CLPS and enabling the expression of our target genes. In the absence of glucose, GlcT remains phosphorylated; thus, it remains unbound from the RAT sequence, keeps the terminator accessible, and stops T7 RNA polymerase mRNA translation. Additionally, without the formation of HPrSerP-CcpA complexes, lacI is transcribed, producing the lac repressor protein (LacI). LacI blocks transcription of T7 RNA polymerase, PNLIP, and CLPS, effectively shutting down gene expression in low-glucose conditions.

We chose to add His-6 and HA tags, respectively, to the PNLIP and CLPS coding sequences with for protein purification purposes. Additionally, as these proteins are not naturally secreted by our selected chassis, we also flanked our coding sequences with signal peptide coding sequences (described in detail later in this page) so they are flagged for secretion from the cell. However, during the time of design, we were unsure as to which conformation or order of the signal peptide, tag, and coding sequence is most optimal for our intended design. Thus, we synthesized two variations of each coding sequence with its signal peptides in order to determine which was more optimal in our intended design. These two variations are: signal peptide – tag – coding sequence versus signal peptide – coding sequence – tag.

Given the various components of our design and the high variability we sought in our constructs to permit ease of testing and determination of more optimal part conformations, we decided to develop two “storage plasmids” using Gibson Assembly, the more effective method for combining various inserts into one plasmid backbone. We used pBS1C for our plasmid backbone and intended to digest certain parts and re-ligate to form constructs for testing. Our storage plasmids, distinguished by their names “A” and “B” are as described briefly below:

Gibson Plasmids

| Gibson A Plasmid | Gibson B Plasmid |
| --- | --- |
| * 1A: signal peptide - His6 - PNLIP * 2A: signal peptide - HA - CLPS * 3A: T7RNAP * 4A: glucose-metabolite binding site | * 1B: signal peptide - PNLIP - His6 * 2B: signal peptide - CLPS - HA * 3B: RNA anti-terminator (RAT) * 4B: glucose-metabolite binding site |

![first-circuit-design](https://static.igem.wiki/teams/5178/circuit-diagrams/allpartscircuits.png)

**Figure 2.** Diagrams depicting each of the components described above for the Gibson A plasmid and the Gibson B plasmid.

## Part Justifications

### Part 1: Lipase ([BBa\_K5178012](https://parts.igem.org/Part%3ABBa_K5178012))

*Note: As indicated above, we synthesized two versions of this part (Part 1A and Part 1B). Part 1A contained a sequence with the His6 Tag after the signal peptide and before the lipase coding sequence, whereas Part 1B contained a sequence with the His6 tag after the lipase coding sequence. These two iterations allowed us to determine which conformation would permit more efficient secretion and protein folding of the lipase enzyme.*

* PT7lac Promoter: The PT7lac promoter is a chimeric part proven to function in *B. subtilis* and was engineered as a part of an inducible promoter system involving the lac repression protein and T7 RNA polymerase (T7RNAP)[[1]](#citation1). In the presence of LacI, transcription is repressed at PT7lac, minimizing leakiness in the expression of the gene of interest. When no LacI is present, the polymerase binds to this promoter region and actively initiates transcription[[2]](#citation2).
* RiboJ: Insulation with RiboJ increases gene expression at RNA and protein levels[[3]](#citation3). Two parts comprise RiboJ: the sTRSV ribozyme and a 23-nt hairpin. The self-cleaving mechanism of sTRSV causes the remaining sequence to form a terminal hairpin[[3](#citation3),[4]](#citation4). RiboJ shields downstream transcription from upstream factors4, providing stability and increased accuracy to gene transcription. *B. subtilis* is often considered to be leaky in inducible promoter systems and controlling gene transcription is not always accurate; RiboJ is one part that serves to minimize this leakiness[[1]](#citation1).
* MF001 RBS: This ribosome binding site is commonly-used in and optimized for *B. subtilis*. It has precedence in the literature from which the circuit and construct was inspired[[1]](#citation1).
* Human pancreatic lipase gene (PNLIP): As this is the main enzyme lacking in Exocrine Pancreatic Insufficiency (EPI) patients[[5](#citation5),[6]](#citation6), the main goal of our project was to engineer bacteria to secrete this enzyme as an alternative enzyme replacement therapy[[7]](#citation7). Thus, this coding sequence, codon-optimized for *B. subtilis*, permits expression of the protein.
* Lipase signal peptide: As lipase is not naturally secreted from *B. subtilis*, the protein must be tagged with a signal peptide to be secreted from the cell. The signal peptide allows for the protein to be flagged for secretion. The sequence for the signal peptide was determined through reverse translation from the amino acid sequence of a signal peptide with similar function identified in literature[[8](#citation8),[9]](#citation9).
* His6 Tag: The His6 tag allows for the protein to be purified using immobilized metal affinity chromatography (IMAC) due to its high affinity for immobilized metals which provides a simpler purification compared to other methods[[10]](#citation10). The His6 tag also allows us to use anti-His6 antibodies to test for presence rather than anti-lipase antibodies which allows us more financial flexibility.
* B0015 Terminator: This terminator is commonly used in and optimized for *B. subtilis*. It has precedence in the literature from which the circuit and construct was inspired[[1]](#citation1).

### Part 2: Procolipase ([BBa\_K5178006](https://parts.igem.org/Part%3ABBa_K5178006))

*Note: Similar to Part 1, we synthesized two versions of this part (Part 2A and Part 2B) as well. Part 2A contained a sequence with the HA Tag after the signal peptide and before the procolipase coding sequence, whereas Part 2B contained a sequence with the HA Tag after the procolipase coding sequence. These two iterations allowed us to determine which conformation would permit more efficient secretion and protein folding of the procolipase enzyme, which then becomes colipase.*

* PT7lac Promoter: The PT7lac promoter is a chimeric part proven to function in *B. subtilis* and engineered as a part of an inducible promoter system involving the lac repression protein and T7RNA polymerase[[1]](#citation1). In the presence of LacI, transcription is repressed at PT7lac, minimizing leakiness in the expression of the gene of interest. When no LacI is present, the polymerase binds to this promoter region and actively initiates transcription[[2]](#citation2).
* RiboJ: Insulation with RiboJ increases gene expression at RNA and protein levels[[3]](#citation3). Two parts comprise RiboJ: the sTRSV ribozyme and a 23-nt hairpin. The self-cleaving mechanism of the ribozyme causes the remaining sequence to form a terminal hairpin[[3](#citation3),[4]](#citation4). RiboJ shields downstream transcription from upstream factors[[4]](#citation4), providing stability and increased accuracy to gene transcription. *B. subtilis* is often considered to be leaky in inducible promoter systems and controlling gene transcription is not always accurate; RiboJ is one part that serves to minimize this leakiness[[1]](#citation1).
* MF001 RBS: This ribosome binding site is commonly-used in and optimized for *B. subtilis*. It has precedence in the literature from which the circuit and construct was inspired[[1]](#citation1).
* Pancreatic procolipase (CLPS): In the presence of bile salts (which are prevalent in the digestive system), lipase requires a cofactor (colipase) to bind to and catabolize lipid macromolecule structures[[11]](#citation11). Colipase is initially secreted as procolipase, which is then cleaved and transformed into colipase[[12]](#citation12). Thus, this coding sequence is crucial for procolipase to be expressed.
* Procolipase signal peptide: As procolipase is not naturally secreted from *B. subtilis*, the protein must be tagged with a signal peptide to be secreted from the cell. The signal peptide allows for the protein to be flagged for secretion. The sequence for the signal peptide was determined by reverse translation of the nucleotide sequence of a signal peptide with similar function identified in literature[[8](#citation8),[9]](#citation9).
* HA Tag: The HA tag permits efficient and simple affinity purification of the tagged protein. Additionally, the HA tag allows for the protein’s presence to be detected using anti-HA antibodies[[13]](#citation13), which are a much lower financial burden than anti-procolipase antibodies.
* B0015 Terminator: This terminator is commonly used in and optimized for *B. subtilis*. It has precedence in the literature from which the circuit and construct was inspired[[1]](#citation1).

### Composite Part 3: T7RNAP and Terminator/Anti-terminator Sequence for Glucose-Mediated Switch Mechanism

#### Part 3A ([BBa\_K5178017](https://parts.igem.org/Part%3ABBa_K5178017)

*Note: Part 3a has two components, Part 3A.1 and Part 3A.2. Part 3A.2 will be used in the final construct, whereas Part 3A.1 was synthesized purely for testing purposes.*

* ##### Part 3A.1 ([BBa\_K5178014](https://parts.igem.org/Part%3ABBa_K5178014))

  + P43 Promoter: The P43 promoter is a strong, constitutive active promoter. It has been optimized for *B. subtilis*[[14]](#citation14).
  + Lac Operator: The lac operator is the sequence at which LacI binds to prevent transcription in the lac operon mechanism, first described in E. coli[[15]](#citation15). It has been demonstrated to function in *B. subtilis* in literature[[16]](#citation16).
  + MF001 RBS: This ribosome binding site is commonly used in and optimized for *B. subtilis*. It was referenced commonly in the literature from which the circuit and construct was inspired[[1]](#citation1).
* ##### Part 3A.2 ([BBa\_K5178015](https://parts.igem.org/Part%3ABBa_K5178015))

  + T7RNAP gene: The T7 RNA polymerase gene (“T7RNAP gene”) is derived from bacteriophage T7[[17]](#citation17). T7RNAP has a high and specific processivity, with maximal expression of the transcribed gene(s) of interest[[1](#citation1),[17]](#citation17). Additionally, T7RNAP is highly specific toward the T7 promoter[[17]](#citation17), preventing unwanted transcription of other genes in our device with non-T7 promoters. It has been shown that T7RNAP binds strongly to PT7lac and is effectively repressed by LacI[[1]](#citation1), reinforcing our inducing mechanism.
  + B0015 Terminator: This terminator is commonly used in and optimized for *B. subtilis*. It has precedence in the literature from which the circuit and construct was inspired[[1]](#citation1).

#### Part 3B ([BBa\_K5178000](https://parts.igem.org/Part%3ABBa_K5178000))

*Note: will replace Part 3A.1 to create a composite part ([BBa\_K5178016](https://parts.igem.org/Part%3ABBa_K5178016))*

* Lac Operator: The lac operator is the sequence at which LacI binds to prevent transcription in the lac operon mechanism, first described in E. coli[[15]](#citation15). It has been proven to function in *B. subtilis*. in literature[[16]](#citation16).
* PptsG Promoter: The PptsG promoter is a native promoter of *B. subtilis* that is utilized before the ptsG gene within the phosphotransferase system. This is a strong promoter that contains a RAT sequence and a terminator sequence, both of which are used in a mechanism that responds to extracellular glucose (as explained below)[[18]](#citation18).
* RAT Terminator/Anti-Terminator: The RAT sequence and the terminator sequence both follow the PptsG promoter. The RAT sequence forms a mRNA anti-terminator structural hairpin that exposes the terminator sequence so that the following PptsG promoter and the respective coding sequence are inhibited. When there is extracellular glucose present, the phosphotransferase system is activated, and a downstream molecule (phosphorylated GlcT) becomes dephosphorylated. The dephosphorylated GlcT will bind to the RAT sequence, causing a conformational change so that the terminator is inaccessible, and the promoter sequence is available. This will lead to the transcription of the subsequent coding sequence[[19]](#citation19).
* MF001 RBS: This ribosome binding site is commonly used in and optimized for *B. subtilis*. It was referenced commonly in the literature from which the circuit and construct was inspired[[1]](#citation1).

### Composite Part 4: Cre-site Mechanism for Glucose-Mediated Switch Mechanism

*Note: Two versions of this part were synthesized to determine which was more efficient. Part 4A sourced the sequence of the amyE cre site from a database, and Part 4B sourced the sequence of the amyE cre site from literature. GFP, flanked by restriction enzymes, comprised Part 4c; this part was used only to test the relative fluorescent expression of Parts 4A and 4B, and it did not become part of our final construct.*

* 4A: [BBa\_K5178001](https://parts.igem.org/Part%3ABBa_K5178001)
* 4B: [BBa\_K5178002](https://parts.igem.org/Part%3ABBa_K5178002)

* P43 Promoter: The P43 promoter is a strong, constitutive active promoter. It has been optimized for *B. subtilis*[[14]](#citation14).
* amyE cre site: Utilization of the carbon catabolite repression (CCR) pathway in *B. subtilis* allows for a glucose-inducible circuit[[20]](#citation20). Through the uptake of glucose, the protein HPr becomes phosphorylated and binds to Carbon Control Protein A (CcpA)[[21]](#citation21). The HPrSerP-CcpA complex then binds to catabolite responsive elements (cre sites) of DNA sequences[[22]](#citation22). Cre sites can be located upstream or downstream relative to the promoter, and they activate or inhibit gene transcription, respectively[[21]](#citation21). The amyE cre site was selected from over 50 known cre sites from the *B. subtilis* genome for its high affinity and close distance of +4 bp to the TSS of the gene of interest[[21]](#citation21). The amyE cre site is bound and transcription is strongly repressed when glucose is present.
* MF001 RBS: This ribosome binding site is commonly used in and optimized for *B. subtilis*. It was referenced commonly in the literature from which the circuit and construct was inspired[[1]](#citation1).
* LacI gene: lacI is a well-characterized gene first described in E. coli that encodes for the lactose repressor protein (LacI). LacI is a DNA-binding transcription factor that strongly represses transcription[[23]](#citation23). LacI was expressed successfully in *B. subtilis* as a part of the inducible promoter system[[1]](#citation1) and serves to repress T7RNAP expression in our device system by binding to our incorporated lac operator sequence.
* B0015 Terminator: This terminator is commonly used in and optimized for *B. subtilis*. It has precedence in the literature from which the circuit and construct was inspired[[1]](#citation1).
* GFP: One of the most common fluorescent markers used in analyzing expression strength, GFP helps us to assess the two cre site sequences and determine which is more effective. The sequence[[24]](#citation24) was codon-optimized for *B. subtilis*.

## Testing Plan

Gibson assembly permits high modularity of constructs, as we embedded various restriction sites into our Gibson sequences. Using traditional assembly methods (restriction digest and ligation), our design permits us to assemble the following constructs for testing:

![Testing-constructs](https://static.igem.wiki/teams/5178/circuit-diagrams/image-46.webp)

**Figure 3.** Testing constructs to be assembled after Gibson Assembly. These constructs were planned to be assembled using traditional assembly methods.

After testing the various combinations illustrated above, we were planning to determine the most optimal construct based on which lipase part (1A or 1B) and which procolipase part (2A or 2B) resulted in the highest yield of enzymes. We aimed to identify the composite parts that permitted the highest secretion yield through Western Blots of the supernatant after centrifuging a culture containing the transformed bacteria. We also planned to use the Cayman Lipase Activity Assay Kit to ensure that the protein folding and activity of the secreted lipase was as desired. The optimal parts were intended to be combined to produce a 1 + 2 + 3A construct (pBS1C-PNLIP-CLPS-T7RNAP) to test the secretion of enzymes initially before implementing the glucose-mediated switch mechanism. If this was successful, we were planning to then incorporate components of the glucose-mediated switch mechanism to determine how this affects the timescale of production of the enzymes.

## Build

We were successfully able to assemble our Gibson B plasmid using Gibson Assembly methods. Our sequenced plasmid returned with all four inserts within the plasmid. For our Gibson A plasmid, we were successfully able to insert our 1A part, but unable to successfully insert the rest. After numerous unsuccessful attempts, we modified our design plan to focus on using our 1B and 2B conformations. After consultation with experts conducting research regarding signal peptides, they validated the 1B and 2B conformations, with the purification tag placed after the coding sequence.

## Redesign

Due to our obstacles with inserting part 3A, which is crucial for expression of lipase and procolipase by the Gibson B plasmid, we eliminated our Gibson A plasmid and focused on inserting part 3A to permit expression of the enzymes in the Gibson B plasmid.

We attempted to digest between parts 2B and 3B to ligate 3A into the desired location. However, after facing many obstacles and numerous unsuccessful attempts with various ligation kits and methods, we decided to approach an alternative plan.

We designed and ordered parts 1B and 2B modified with constitutive promoters (P43 and Pveg, two strong promoters used in *B. subtilis*) as opposed to the T7 RNA polymerase induced promoter used previously. We renamed these parts 1C and 2C. This permits the expression of lipase and procolipase independently, and thus part 3A is not necessary. Though this hinders our ability to test the glucose-switch mechanism as it was depending on the T7 RNAP polymerase, it allows us to overcome the obstacles faced with ligating 3A, a part nearly 3 kb long, into the construct. Overcoming these obstacles permits us to test the synthesis, expression, and secretion of lipase and procolipase from *B. subtilis*.

![constitutive-circuit-design](https://static.igem.wiki/teams/5178/circuit-diagrams/constcircuits.png)

**Figure 4.** Constitutive parts for expression and secretion of lipase and procolipase.

After testing the various combinations illustrated above, we were planning to determine the most optimal construct based on which lipase part (1A or 1B) and which procolipase part (2A or 2B) resulted in the highest yield of enzymes. We aimed to identify the composite parts that permitted the highest secretion yield through Western Blots of the supernatant after centrifuging a culture containing the transformed bacteria. We also planned to use the Cayman Lipase Activity Assay Kit to ensure that the protein folding and activity of the secreted lipase was as desired. The optimal parts were intended to be combined to produce a 1 + 2 + 3A construct (pBS1C-PNLIP-CLPS-T7RNAP) to test the secretion of enzymes initially before implementing the glucose-mediated switch mechanism. If this was successful, we were planning to then incorporate components of the glucose-mediated switch mechanism to determine how this affects the timescale of production of the enzymes.

## Test

Due to the various obstacles faced with previous ligations, our timeline was delayed from that which was expected. However, we were able to test our constitutive parts with a lipase assay and concluded that lipolytic activity was present. We also tested our constitutive parts for their expression of lipase and procolipase using SDS-PAGE, and can reasonably conclude that we have expressed lipase. Our gel was more difficult to interpret for secretion of procolipase, but based on sequencing data, we believe that procolipase is also expressed. Moving forward, we will continue testing our construct by reattempting the lipase assay with suitable controls and performing a Western Blot using our His6 and HA tags to confirm expression of lipase and procolipase in *E. coli* and *B. subtilis.*

We also tested our glucose switch separately and determined that, as expected, GFP fluorescence was reduced in the presence of glucose. However, since the excitation peak was not at GFP's typical excitation peak, it appears that glucose is influencing cellular physiology in unanticipated ways, and in particular could be triggering autofluorescence. Our future steps include further testing to explain these changes.

After confirming enzyme secretion and conducting further testing on our glucose switch, we plan to integrate both components in order to have a fully-functioning circuit that allows our bacteria to express lipase and procolipase in response to glucose input.

## Safety Parts and Engineering

Our device also contains a biocontainment mechanism. The parts and engineering for this mechanism is described in the [Safety page.](https://2024.igem.wiki/virginia/safety)

## References

## Click to expand

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[[2]](#2) Dubendorf, J. W.; Studier, F. W. Controlling Basal Expression in an Inducible T7 Expression System by Blocking the Target T7 Promoter with Lac Repressor. J. Mol. Biol.  **1991,** *219* (1), 45–49. [https://doi.org/10.15403/jgld-5005.](https://pubmed.ncbi.nlm.nih.gov/38386889/)

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[[4]](#4) Lou, C.; Stanton, B.; Chen, Y.-J.; Munsky, B.; Voigt, C. A. Ribozyme-Based Insulator Parts Buffer Synthetic Circuits from Genetic Context.Nat. Biotechnol. **2012,** *30* (11), 1137–1142.
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[[5]](#5)  Lipase Information | Mount Sinai -
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[[8]](#8) Fu, G.; Liu, J.; Li, J.; Zhu, B.; Zhang, D. Systematic Screening of Optimal Signal Peptides for Secretory Production of Heterologous Proteins in Bacillus Subtilis.J. Agric. Food Chem. **2018,** *66* (50), 13141–13151.
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[[9]](#9) Yang, H.; Qu, J.; Zou, W.; Shen, W.; Chen, X. An Overview and Future Prospects of Recombinant Protein Production in Bacillus Subtilis.Appl. Microbiol. Biotechnol. **2021,** *105* (18), 6607–6626.
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[[10]](#10) Bornhorst, J. A.; Falke, J. J. [16] Purification of Proteins Using Polyhistidine Affinity Tags.Methods Enzymol. **2000,** *326* , 245–254.

[[11]](#11) van Tilbeurgh, H.; Sarda, L.; Verger, R.; Cambillau, C. Structure of the Pancreatic Lipase–Procolipase Complex. Nature. **1992,** *359* (6391), 159–162.
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[[12]](#12) Larsson, A.; Erlanson-Albertsson, C. The Effect of Pancreatic Procolipase and Colipase on Pancreatic Lipase Activation. Biochim. Biophys. **1991,** *1083* (3), 283-288.
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[[14]](#14) Part:BBa K143013. Part:BBa K143013 - parts.igem.org.
[https://parts.igem.org/Part:BBa\_K143013.](https://parts.igem.org/Part%3ABBa_K143013.) (accessed 2024-07-22).

[[15]](#15) Gilbert, W.; Maxam, A. The Nucleotide Sequence of the Lac Operator. Proc. Natl. Acad. Sci. U. S. A.  **1973,** *70* (12 Pt 1-2), 3581–3584.

[[16]](#16) Yansura, D. G.; Henner, D. J. Use of the Escherichia Coli Lac Repressor and Operator to Control Gene Expression in Bacillus Subtilis. Proc. Natl. Acad. Sci. U. S. A.  **1984,** *81* (2), 439–443.

[[17]](#17) Borkotoky, S.; Murali, A. The Highly Efficient T7 RNA Polymerase: A Wonder Macromolecule in Biological Realm. Int. J. Biol. Macromol. **2018,** *118*, 49-56.
<https://doi.org/10.1016/j.ijbiomac.2018.05.198.>

[[18]](#18) Schmalisch, M. H.; Bachem, S.; Stülke, J. Control of the Bacillus Subtilis Antiterminator Protein GlcT by Phosphorylation. J. Biol. Chem. **2003,** *278* (51), 51108–51115.
<https://doi.org/10.1074/jbc.M309972200.>

[[19]](#19) Schilling, O.; Langbein, I.; Müller, M.; Schmalisch, M. H.; Stülke, J. A Protein‐dependent Riboswitch Controlling ptsGHI Operon Expression in Bacillus Subtilis: RNA Structure Rather than Sequence Provides Interaction Specificity. Nucleic Acids Res. **2004,** *32* (9), 2853–2864.
<https://doi.org/10.1093/nar/gkh611.>

[[20]](#20) Tavares, L. F.; Ribeiro, N. V.; Zocca, V. F. B.; Corrêa, G. G.; Amorim, L. A. S.; Lins, M. R. C. R.; Pedrolli, D. B. Preventing Production Escape Using an Engineered Glucose-Inducible Genetic Circuit. ACS Synth. Biol. **2023,** *12* (10), 3124–3130.
<https://doi.org/10.1021/acssynbio.3c00134.>

[[21]](#21) Marciniak, B. C.; Pabijaniak, M.; de Jong, A.; Dűhring, R.; Seidel, G.; Hillen, W.; Kuipers, O. P. High- and Low-Affinity Cre Boxes for CcpA Binding in Bacillus Subtilis Revealed by Genome-Wide Analysis. BMC Genomics. **2012,** *13* (1), 401.
<https://doi.org/10.1186/1471-2164-13-401.>

[[22]](#22) Jones, B. E.; Dossonnet, V.; Küster, E.; Hillen, W.; Deutscher, J.; Klevit, R. E. Binding of the Catabolite Repressor Protein CcpA to Its DNA Target Is Regulated by Phosphorylation of Its Corepressor HPr\*. J. Biol. Chem. **1997,** *272* (42), 26530–26535.
<https://doi.org/10.1074/jbc.272.42.26530.>

[[23]](#23) Escherichia coli K-12 substr.MG1655 lacI.
<https://biocyc.org/gene?orgid=ECOLI&id=EG10525.>(accessed 2024-07-22).

[[24]](#24) LLC, G. B.GFP Sequence and Map.
[https://www.snapgene.com/plasmids/fluorescent\_protein\_genes\_and\_plasmids/GFP .](https://www.snapgene.com/plasmids/fluorescent_protein_genes_and_plasmids/GFP%20.) (accessed 2024-07-23).

# **Experiments**

[Click to read content](#Experiments)

![](https://static.igem.wiki/teams/5178/headers/barry/barry-gel.webp)

* [Experimental Workflow](#Experimental-workflow)
* [Protocols](#Protocols)

## Experimental Workflow

### Designing Bliss

BLISS’s entire genetic circuit consists of over 7000 base pairs, meaning it could not be
synthesized in entirety due to size and
monetary constraints. As a workaround, we broke our circuit into four unique parts, each
under 3000 base pairs.
Variations of the four parts were also created and denoted as “A” or “B.”
Table 1 (below) describes what each number-letter combination represents.

| Part Name | Description |
| --- | --- |
| 1A | signal peptide + His6 tag + human pancreatic lipase gene |
| 1B | signal peptide + human pancreatic lipase gene + His6 tag |
| 2A | signal peptide + HA tag + pancreatic procolipase gene |
| 2B | signal peptide + pancreatic procolipase gene + HA tag |
| 3A | T7RNA polymerase gene |
| 3B | RNA terminator/antiterminator sequence |
| 4A | carbon catabolite responsive element binding site (from database) |
| 4B | carbon catabolite responsive element binding site (from publication) |

**Table 1.** Number-letter names for each part and their
corresponding significance. More information about each part can be found under the [Engineering page](https://2024.igem.wiki/virginia/engineering) of our wiki.

Modifications—such as rearrangements of genes, signal peptides, and purification tags in
parts 1A, 2A, 1B, and 2B—were made to improve chances of the circuit functioning as
intended. Parts 4A and 4B serve identical functions—to bind carbon catabolite complex,
HPrSerP-CcpA, and halt expression of lacI—however, literature and database coding sequences
conflicted with each other. Both variations were synthesized, and the functionality of each
will be tested in the future with a fluorescence assay.

All parts were ordered as gene fragments from International DNA Technologies (IDT).
Each part was synthesized with 50 base pair Gibson Assembly overhangs meticulously designed
to include internal restriction enzyme cut sites. This design allows all parts to be
assembled in one step with Gibson Assembly into two “storage plasmids,” designated “storage
plasmid A” and “storage plasmid B,” where storage plasmid A contains all four “A parts” and
storage plasmid B contains all four “B parts” (Figure 1). From these storage plasmids,
individual parts can be isolated with the internal restriction enzyme cut sites. This
modular design allows us to create entirely new and customized constructs suitable for the
various assays needed to test functionality of individual parts.

![Figure of A and B plasmids](https://static.igem.wiki/teams/5178/gibson-a-and-b.png)

**Figure 1.** Fully assembled Gibson A and Gibson B storage
plasmids. A. Gibson A storage plasmid with parts: 1A, 2A, 3A, and 4A. B. Gibson B storage
plasmid with parts: 1B, 2B, 3B, and 4B.

### Gibson Assembly

Gibson Assembly was chosen to construct our storage plasmids because it allowed us to
construct each plasmid in one reaction, saving time and resources. Plasmid pBS1C was chosen
as the backbone for our storage plasmids because it contains genes encoding for both
ampicillin and chloramphenicol resistance. pBS1C was also selected because it can be
integrated into the chromosomal DNA of *B. subtilis*, preventing the plasmid from being
lost or excised. Another unique feature of pBS1C is that it contains a coding sequence for
red fluorescent protein (RFP). When RFP is cut out from the backbone and DNA parts are
successfully cloned in, transformed *E. coli* colonies will appear white. Any red
colonies present after transformation are immediate feedback cloning wasn’t successful.

During our first attempt at Gibson Assembly, we sought to create both A and B
storage plasmids. After digesting pBS1C with EcoRI and PstI to remove the RFP, we combined
the digested pBS1C with all A parts and all B parts in two separate reactions. Gibson
Assembly products were transformed into NEB high competency *E. coli* DH5α cells, then
plated on selective LB-ampicillin medium. Several resulting colonies on each plate were
grown in overnight cultures, miniprepped, then analytically digested and screened on an
agarose gel. The gel results, viewable on the [Results page](https://2024.igem.wiki/virginia/results), weren’t exactly
what we expected, however, one A and one B colony were selected for sequencing. Sequencing
performed by GeneWiz (Azenta Life Science) revealed parts 1A and 1B had been successfully
cloned into pBS1C. The remaining parts (i.e. 2A, 2B, 3A, 3B, 4A, and 4B) were not assembled
because the 3’ sequences of 1A and 1B had a six base pair region of complementarity with the
cut site created by PstI digesting pBS1C. This allowed parts 1A and 1B to ligate to the
backbone instead of the 5’ Gibson sequences of the consecutive 2A and 2B parts. A more
detailed explanation of this outcome can be found within the [Results page](https://2024.igem.wiki/virginia/results) of our wiki.

While cloning parts 1A and 1B into pBS1C was a success, they are unusable without
part 3A: T7 RNA Polymerase, which serves to activate the promoters regulating the lipase and
colipase genes. In following weeks, we attempted several ligations to create constructs
containing 1A, 2A, and 3A. A more in-depth explanation of the ligation process is found
under the [Ligation section](#ligation) of this page. Alongside ligation, we also reattempted to create
storage plasmids A and B using Gibson Assembly. Eventually, we were able to fully assemble
storage plasmid B containing parts 1B, 2B, 3B, and 4B. To date, we have not been able to
assemble storage plasmid A.

One Gibson Assembly attempt, we utilized an inverse PCR technique on our plasmid
containing part 1A. We designed PCR primers to amplify and linearize the entire plasmid in a
way that exposed the 3’ Gibson Assembly sequence of part 1A so that we could add in parts
2A, 3A, and 4A. The resulting constructs were digested and run on an agarose gel. Several
bands were observed; however, the quantity and size of the bands did not align with
anticipated results. For this reason, this attempt was ultimately considered unsuccessful.
We have not reattempted Gibson Assembly for storage plasmid A due to the redesign of parts 1
and 2.

Several times, we attempted to clone part 3A into our pBS1C-1A and pBS1C-2A
constructs with traditional ligation methods. With time running out before the Jamboree, we
decided to redesign parts 1B and 2B so that they are constitutively active, rather than
dependent on part 3A to activate. These new parts have been named parts 1C and 2C. Each
contains the same coding sequences for lipase and procolipase, however, the promoter
controlled by T7 RNA Polymerase was swapped with a constitutively active one. The new parts
have yet to arrive, and the team anticipates attempting Gibson Assembly with them soon.

### Ligation

BLISS’s modular design allows individual parts to be removed from storage plasmids with
restriction enzymes. Enzyme cut sites were strategically placed so that we could cut out and
ligate parts together, creating new constructs with specified functions. We prioritized
creating constructs 1A + 2A + 3A and 1B + 2B + 3A, as these combinations of parts would
allow us to test for enzyme secretion in transformed *B. subtilis*.

Each ligation began with digesting the storage plasmid containing the parts we
needed and digesting our synthesized stock solution of part 3A with the same restriction
enzymes. After digestion, the DNA was run through an agarose gel, and DNA bands of interest
were gel extracted. Once isolated, the DNA was ligated on several occasions with different
ligases including T4 and T7 DNA ligases, as well as the NEB Quick Ligation Kit. To increase
chances of success, the ligation reaction mixture was incubated at varying times and
temperatures. After the ligation reactions were transformed into competent *E. coli*,
analytical digests of transformed colonies were performed. No positive results were
obtained.

In-agarose ligation was also attempted. Different than traditional ligation,
in-agarose ligation involves ligating digested DNA directly from an agarose gel—no
extraction necessary. After transformation of the ligation product, no colonies (with the
exception of a positive control) were yielded.

After several attempts and many rounds of troubleshooting, the team has yet to
successfully create a new construct through ligation. Efforts remain ongoing to create a
construct capable of secreting enzymes lipase and procolipase.

### Biocontainment Switch Mechanism

We planned to assemble the three parts – xylose induced antitoxin, temperature sensitive
component, and xylose repressor – with traditional assembly. We digested the pHY300PLK
backbone, the xylose induced antitoxin part, and the xylose repressor part and ran through a
gel. We then gel extracted and attempted ligation, transformed into *E. coli*, and
plated on 1%, 2%, and 4% xylose-amp plates. We attempted this twice with both times failing.

Thus, we re-evaluated our parts and concluded that a design error may be causing
failed ligation. Therefore, we designed PCR primers with flanking Gibson overhangs with the
goal of adding these sequences to the part and then reattempting assembly via the Gibson
assembly method. We have attempted PCR with the new primers which we verified on a gel, and
it appears to be successful. Additionally, we digested the pHY300PLK backbone in preparation
for Gibson assembly which we then ran through a gel and extracted. We plan to attempt Gibson
assembly in the following week and if successful, we will transform into *B. subtilis*
and perform viability assays.

## Protocols

This PDF contains all protocols used by this year's Virginia iGEM team.

# **Results**

[Click to read content](#gibson-assembly)

![](https://static.igem.wiki/teams/5178/headers/barry/experiments-no-words.webp)

* [Assembly](#gibson-assembly)
* [Fluorescence Assay](#fluorescence-assay)
* [Lipase Updates](#lipase-updates)
* [Glucose Updates](#glucose-updates)

## Assembly

### Gibson Plasmid A

![](https://static.igem.wiki/teams/5178/results/screenshot-2024-10-02-at-5-29-47-am.png)

**Figure 1.** Gibson Plasmid A containing parts 1A (pancreatic lipase gene), 2A (pancreatic colipase gene), 3A (T7 RNA polymerase gene), and 4A (cre binding sites + lacI gene).

On July 22nd, we attempted Gibson Assembly with our “A” parts, specifically 1A (lipase), 2A (colipase), 3A (T7 RNA Polymerase), and 4A (cre-binding site component) (Figure 1). After combining the inserts with the vector backbone (the pBS1C plasmid, digested from EcoRI to PstI) and the Gibson HiFi Master Mix (New England Biolabs, E2611S), we transformed NEB® 5-alpha Competent *E. coli* (High Efficiency) (New England Biolabs, C2987H) NEB Competent *E. coli* cells with our Gibson Assembly plasmid. The pBS1C backbone permits red-white screening to ensure that the transformation was successful. The digest from EcoRI to PstI should remove the RFP gene from the backbone, and thus any recircularized plasmids should appear white. Any red/pink plasmids are undigested plasmid and thus do not contain the desired inserts.

On July 23rd, we observed growth of one white colony on the LB-ampicillin plate (Figure 1C). This indicates that the Gibson Assembly may have been successful, as the cells need a recircularized vector containing the ampicillin resistance gene to successfully grow on the selective media. We established an overnight culture with this colony, and then mini-prepped the DNA (Zymo Research, ZymoPURE Plasmid Miniprep Kit, D4211). The DNA was used for analytical digests in the laboratory, but was also sent to GenScipt for sequencing.

![](https://static.igem.wiki/teams/5178/results/screenshot-2024-10-02-at-4-52-58-am.png)

**Figure 2.**  Gibson Assembly constructs assembled into NEB super competent DH5A. All colonies grown on LB-ampicillin medium. A. Transformed with pUC19 control vector. Several colonies present. B. Transformation with no insert. No colonies present. C. Transformation with Gibson Assembly A constructs. 1 white colony present (red box). D. Transformation with Gibson Assembly B constructs, 3 white colonies present (red box).

![](https://static.igem.wiki/teams/5178/results/screenshot-2024-10-02-at-4-54-44-am.png)

**Figure 3.**  A. 1% agarose gel with ten lanes of linearized DNA. Lanes (from left to right) loaded with: Quick-Load® 1 kb Plus DNA Ladder (New England Biolabs, N0469S), blank, pBS1C, blank, A1: Gibson Assembly A colony 1, blank, B1: Gibson Assembly B colony 1, B2: Gibson Assembly B colony 2, B3: Gibson Assembly B colony 3, blank. All DNA samples were digested with SmaI. B. Expected results for gel shown in A when DNA is fully digested with SmaI.

![](https://static.igem.wiki/teams/5178/results/screenshot-2024-10-02-at-5-35-17-am.png)

**Figure 4.**  Gibson Assembly (from July 22nd, 2024) data and hypothesis. A. pBS1C with part “1A” corresponding to human pancreatic lipase (PNLIP). B. Diagram hypothesizing how only part 1A was inserted during Gibson Assembly. The end Gibson sequence of 1A was able to bind to an overhang created by a PstI cut site.

We attempted multiple additional Gibson Assembly methods to successfully assemble all four of our desired parts in one construct. These included various methods, such as inverse PCR of the pBS1C-1A plasmid, reattempting Gibson Assembly with sequential addition of inserts after incubation periods, and adding the vector last to permit attachment of inserts first, but none of these were successful.

### Gibson Plasmid B

Throughout the month of July, we performed many attempts at Gibson Assembly with our “B” parts, but these proved to be unsuccessful. On July 29th, we reattempted Gibson Assembly with the “B” parts, specifically 1B (lipase), 2B (colipase), 3B (terminator/anti-terminator sequence), and 4B (cre-binding site component). After combining the inserts with the vector backbone (the pBS1C plasmid, digested from EcoRI to PstI) and the Gibson HiFi Master Mix ((New England Biolabs, E2611S), we transformed NEB® 5-alpha Competent *E. coli* (High Efficiency) (New England Biolabs, C2987H) with our Gibson Assembly plasmid. As this is the same vector used in the Gibson Plasmid A assembly, we were able to use red-white screening to determine if transformation and assembly were successful.

On July 30th, we observed growth of over 25 colonies on the LB-ampicillin plates. This transformation efficiency and growth was much higher than results seen in previous Gibson Assembly attempts, and thus had high potential for success. We established an overnight culture with this colony, and then miniprepped the DNA (Zymo Research, ZymoPURE Plasmid Miniprep Kit, D4211). The DNA was used for analytical digests in the laboratory, but was also sent to PlasmidSaurus for sequencing.

![](https://static.igem.wiki/teams/5178/results/screenshot-2024-10-02-at-5-07-47-am.png)

**Figure 5.**
Colonies transformed with Gibson B construct (from July 30th, 2024) on an LB-ampicillin plate.

On August 10th, we received our sequencing results from PlasmidSaurus. We were able to successfully clone all four “B” parts into the pBS1C backbone, producing a pBS1C-1B-2B-3B-4B construct.

![](https://static.igem.wiki/teams/5178/results/screenshot-2024-10-02-at-5-31-56-am.png)

**Figure 6.**
Gibson B construct with all four parts: 1B (lipase), 2B (colipase), 3B (RAT-T), and 4b (cre-binding site).

### Inverse PCR

After successfully assembling Gibson plasmid B, we still lacked a plasmid containing all our “A” parts. This plasmid, named “Gibson plasmid A,” needed to include parts 1A, 2A, 3A, and 4A. Instead of starting Gibson Assembly from scratch, we attempted to use an existing plasmid containing part 1A to assemble all A parts into one plasmid. We designed primers (International DNA Technologies) to linearize the plasmid containing 1A in a way that exposed the 3’ Gibson Assembly sequence of part 1A so it could be used in a secondary Gibson Assembly reaction. After PCR amplification with the new primers, we attempted Gibson Assembly (using protocol recommended by New England Biolabs) of Gibson plasmid A on August 12th. Several colonies of transformed *E. coli* JM109 grew, and 18 colonies were selected for screening. The 18 colonies were grown overnight, miniprepped (Zymo Research, ZymoPURE Plasmid Miniprep Kit, D4211), then digested with KpnI. The digested constructs were run on an agarose gel, which was deemed inconclusive due to control bands not aligning with expected band sizes.

### Ligation

With Gibson plasmid B, the team first attempted to make a usable construct by inserting part 3A with traditional ligation. Using restriction enzyme sites designed into the Gibson Assembly sequences, we attempted to create constructs: 1B + 3A, 2B + 3A, and 1B + 2B + 3A. After digesting Gibson plasmid B with a combination of RsrII, PstI, and BspDI, and part 3A with the same enzymes, parts were attempted to be ligated together following New England Biolab’s protocol for T4 DNA Ligase. However, after transforming the ligated constructs, we observed no growth of cells on the plates. Thus, it is likely that the ligation was unsuccessful. We repeated the procedure using different ligases (Hi-T4) and different incubation periods/temperatures (overnight, at room temperature, etc.). We also attempted to perform ligation in-agarose. All attempts at ligation were unsuccessful, and we are still troubleshooting why this may have occurred. It is possible that our 3 kb insert (part 3A) was too large to be inserted into the vector without overburdening the cell. However, we have not yet arrived at any conclusions and need to conduct further testing to do so.

### Gibson 4A and 4B

To test our glucose switch, we needed to isolate parts 4A and 4B, which correspond to two parts with different cre sites under the control of a constitutive promoter. To simplify assays and testing, we decided each part needed a visible reporter to qualitatively confirm function. Green fluorescent protein (GFP) was chosen as a reporter due to its good characterization in fluorescent assays. To insert GFP into parts 4A and 4B, custom PCR primers for parts 4A, 4B, and GFP—each including complementary Gibson overhangs—were ordered and synthesized by Integrated DNA Technologies (IDT). After PCR amplification with the new primers, the parts and GFP (now with Gibson overhangs) were run through a 1% agarose gel to isolate the desired DNA. The bands were then gel extracted (Zymo Research, Zymoclean™ Gel DNA Recovery Kit, D4007). Parts 4A + GFP and 4B + GFP were then assembled with Gibson Assembly, following New England Biolabs’ recommended protocol. After transformation with competent *E. coli* JM109 cells, several colonies were selected for screening. An analytical digest using restriction enzyme, BamHI-HF (New England Biolabs, R3136S), was performed on several colonies. The resulting gel aligned with expected results, suggesting Gibson Assembly was successful (Figure 7). Two colonies were sent to Plasmidsaurus for sequencing.

![](https://static.igem.wiki/teams/5178/results/screenshot-2024-10-02-at-5-14-14-am.png)

**Figure 7.**
Constructs (4A + GFP and 4B + GFP) from Gibson Assembly attempt were digested with restriction enzyme BamHI.

Sequencing revealed that the selected colonies contained the correctly assembled construct (Figure 8). The construct was miniprepped from overnight cultures of the selected colonies (Zymo Research, ZymoPURE Plasmid Miniprep Kit, D4211), then immediately transformed into *B. subtilis* 168. If the parts are working correctly, transformed *B. subtilis* should express green fluorescent protein constitutively, and in the presence of glucose, fluorescence should diminish or disappear. We have yet to observe fluorescence in transformed *B. subtilis* grown in glucose-free medium, but testing remains ongoing.

![](https://static.igem.wiki/teams/5178/results/screenshot-2024-10-02-at-5-20-35-am.png)

**Figure 8.**
Sequencing results from Plasmidsaurus. A. Plasmid with part 4A and GFP. B. Plasmid with part 4B and GFP.

### Biocontainment Switch

Our biocontainment switch contains three unique subparts: a xylose induced antitoxin, a temperature sensitive component, and a xylose repressor. The xylose induced antitoxin corresponds to a coding sequence for toxin MaxE under the control of a xylose-sensitive promoter, the temperature sensitive component corresponds to a coding sequence for antitoxin MazF controlled by a temperature-sensitive RBS, and xylose repressor corresponds to the coding sequence for a xylose operon. We initially designed our parts so they could be assembled through traditional ligation; however, all attempts at ligation have failed. When troubleshooting ligation, we recognized a shortcoming in our design: we neglected to include a six base pair overhang after the last restriction enzyme cut site in each synthesized part. We hypothesize this oversight is the reason our ligations have been unsuccessful, as the lack of extra base pairs prevents the restriction endonuclease from binding and cutting appropriately.

To continue assembling our biocontainment switch, we shifted our method of construction to Gibson Assembly. To do this, we ordered custom PCR primers for each part from International DNA Technologies (IDT). The new primers contain overhangs corresponding to Gibson Assembly sequences. After PCR amplification, the parts will be compatible with Gibson Assembly, and we will be able to construct the kill switch. As of October 2nd, Gibson Assembly has not been completed, but preparative steps are being taken. The team has successfully amplified all parts with the new primers, and we will begin assembly soon.

## Fluorescence Assay

### Parts 4A and 4B

Currently, we possess two kinds of transformed *B. subtilis* 168 containing plasmids with parts 4A and 4B. Each plasmid contains a promoter (regulated by two cre sites), which expresses GFP constitutively. When *B. subtilis* is exposed to glucose in the cellular environment, it transforms glucose into a carbon catabolite, which will bind to the cre sites, interrupting the constitutive promoter’s ability to express GFP. This means, in the absence of glucose, the transformed *B. subtilis* will fluoresce, however, we have yet to observe any fluorescence. We have examined transformed colonies under a fluorescent microscope and fluorescent plate reader to quantify fluorescence. However, we have observed no fluorescence with either machine.

## Lipase Testing Updates

### Lipase Assay

We assembled our desired construct with the constitutive promoters (parts 1C and 2c) into the pBS1C backbone using Gibson Assembly methods. We then transformed this into DH5α *E. coli cells*. Using the transformed cell cultures, we conducted a preliminary assay to detect lipase activity in the DH5α *E. coli*. We identified an average of 18.6 +/- 0.25 times increase between the slope (dAbsorbance/dtime) of each *E. coli* sample compared with its background sample.

![](https://static.igem.wiki/teams/5178/abs510.webp)

**Figure 1.**
Change in Absorbance over time measured to assay for lipase presence and concentration in sample 6. Measured at absorbanec/emission wavelengths of 380/510.

![](https://static.igem.wiki/teams/5178/abs520.webp)

**Figure 2.**
Change in Absorbance over time measured to assay for lipase presence and concentration in sample 6. Measured at absorbanec/emission wavelengths of 390/520.

### Sequencing

These findings showed promise, but we were uncertain if this was due to native lipolytic activity in *E. coli*. To distinguish, we sent a couple of samples in for sequencing. The sequencing results are depicted below.

![](https://static.igem.wiki/teams/5178/sequencing-1.webp)![](https://static.igem.wiki/teams/5178/sequencing-2.webp)

**Figure 3.**
Sequencing results for two experimental constructs assembled to contain human pancreatic lipase (PNLIP) and procolipase (CLPS) in pBS1C backbone.

### SDS-PAGE

One of the samples confirmed 95% match length and 93.5% identity match for CLPS or procolipase and another confirmed 82.14% match length and 100% identity match for CLPS or procolipase. Both had 27% match length and 100% identity match with PNLIP or lipase. However, we were unsure if the lower percentage for lipase was caused by missing basepairs or incorrect sequencing, so we proceeded with an SDS-PAGE after transforming the plasmid into BL21, a higher expression *E. coli* variant, and *B. subtilis* 168, our target chassis.

1. SeeBlue Protein Standard
2. B. subtilis 168-lipase-procolipase-6
3. B. subtilis 168-pBS1C
4. SeeBlue Protein Standard
5. E. coli BL21-lipase-procolipase-10
6. E. coli BL21-lipase-procolipase-9
7. E. coli BL21-lipase-procolipase-6
8. E. coli BL21-pBS1C
9. E. coli BL21 control
10. BioRad Protein Standard

![](https://static.igem.wiki/teams/5178/sds-page.webp)

**Figure 4.**
Coomassie-stained SDS-PAGE gel with experimental samples containing human pancreatic lipase and procolipase expressed in E. coli BL21 and B. subtilis 168, alongside control samples. Lane descriptions are provided above.

We expected to see lipase and procolipase in lanes 2, 5, 6, and 7. Human pancreatic lipase has an expected molecular weight of 47 to 51 kDa. It is seen in our experimental lanes that there is a band of greater intensity between this expected range, and thus it appears that lipase is expressed by the samples in lanes 2, 5, and 6. Procolipase has an expected molecular weight of 12 kDa. However, this region of our gel is difficult to interpret and no significant conclusions can be drawn. Based on our sequencing data, we believe that procolipase is also expressed by the samples.

Our future steps for lipase testing include reattempting the lipase assay with controls to determine the extent to which the results yielded were from native lipolytic activity in *E. coli*. Additionally, we seek to attempt a Western Blot using our His6 and HA tags to confirm the expression of lipase and procolipase in *E. coli* and *B. subtilis*. Additionally, we seek to measure the protein expression within the *B. subtilis* supernatant to confirm enzyme secretion from the cells.

## Glucose Testing Updates

Glucose switch construction was successfully accomplished and transformed in *E. coli*. We incubated half of the transformed cells in glucose-rich media and the other half in glucose-free media. The measurement of GFP fluorescence showed a two-fold repression in the glucose-rich environment, suggesting glucose was inhibiting gene expression.

| Glucose Content | A1 | A2 | B1 | B2 |
| --- | --- | --- | --- | --- |
| Glucose-rich | 2450 | 1948 | 2302 | 2530 |
| Glucose-free | 5215 | 4710 | 4910 | 4995 |
| Ratio | 2.17 | 2.42 | 2.13 | 1.97 |

As demonstrated in the chart, when measuring the strength of green fluorescence (GFP expression) at 395 nm excitation and 510 nm emission, both A and B versions of the cre site sequence showed reduced downstream GFP expression by half in glucose-rich media compared to glucose-free media.
However, the excitation peak was at 335 nm, which is far from GFP's typical 395 nm excitation. 335 nm is a well-known excitation range for autofluorescent molecules. Our initial findings indicate that glucose is influencing *B. subtilis* in ways not previously anticipated, triggering significant changes in other cellular activities, potentially including those responsible for autofluorescence. Thus, our future goals include conducting further research on how glucose impacts the cell's physiology.