Preparation

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)?'.
    Brainstorming our project

    June

    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

    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

    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. coliJ 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

    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

    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

    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

    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

    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

    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

    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

    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

    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

    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. coliJM109
    • 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

    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. coliJM109
      • 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

    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

    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.

    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