Team:NCKU Tainan/Notebook
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Week 1 (6/2 - 6/8)
Wet
Fragment preparation: cmR from pkD3 for can gene knockout and dap gene knockout.
Learning to prepare high CFU heat shock competent cell.
Basic lab skills.
PCR to get tyrP, yebF, CBMB fragment.
Dry
Design and fabricate the first version of microfluidic chip.
Design Wiki page structure.
Do modeling Research and brainstorming about possible modeling topic.
HP
Write news script for NCKU News Agency.
Review Tainan First Senior High School’s feedback.
Week 2 (6/9 - 6/15)
Wet
First attempt can gene knockout and dap gene knockout by electroporation in E. coli MG1655 and E. coli Nissle.
First attempt to ligate each PCR product into pSB1C3.
HP
Contact Genemont and Geneferm companies to entreprise visit.
Week 3 (6/16 - 6/24)
Wet
E. coli MG1655 Δcan::cmR, E. coli Nissle Δcan::cmR, E. coli MG1655 Δdap::cmR, E. coli Nissle Δdap::cmR genotype and phenotype check.
Vector: pSB3K3 transformation and extraction.
Colony PCR to check colony of bacteriocin constructs and tyrP construct.
Use commercial cloning kit to subclone each PCR product into pCR-Blunt II-TOPO and pJET1.2 cloning vector.
HP
Contact tFDA, NHI.
Design and choose our souvenirs.
Week 4 (6/23 - 6/29)
Wet
Kill-switch comparison.
Remove pkD46 from E. coli MG1655 Δcan::cmR and E. coli Nissle Δcan::cmR.
Double digest vector pSB3K3 and Topo-tyrP and further ligation.
Use pJET1.2 to subclone yebF-CBMB.
Dry
Design the second version of microfluidic chip.
Test and calibrate the detector.
HP
Contact with Taiwan Chronic Kidney Disease foundation to join their event on August.
iGEM Collaboration.
Week 5 (6/30 - 7/6)
Wet
IDT fragment preparation and first PCR.
First attempt to construct E. coli Nissle Δcan::FRT.
Sequencing Topo-tyrP, to confirm our tyrP is correct.
Ligation and Transformation for pSB3K3-tyrP into E. coli DH5α.
Ligation and Transformation for pSB3K3-yebF-CBMBs into E. coli DH5α.
Dry
Design Wiki layout.
Design spinner and control rotation speed through Arduino.
Brainstorming IOT part of the hardware.
Model Bacteriocin experiment
HP
Oh My Gut logo design.
Video for wiki.
Genmont Enterprise Visit
Week 6 (7/7 - 7/13)
Wet
Vector: pSB1C3 transformation and extraction.
Second and third overhang extension PCR, double digestion and ligation for pSB1C3-FNR-native RBS-TAL, pSB1C3-FNR-B0034-TAL, pSB1C3-pchR-native RBS-GFP construct.
Extraction for pSB3K3-tyrP, and check by Colony PCR and Double Digestion
Transformation for pFNR-GFP for part characterized.
SDS-PAGE for pSB3K3-tyrP protein expression.
Ligation and Transformation for pSB3K3-yebF-CBMBs into E. coli DH5α.
Dry
Identify cause of spinner vibration.
Model p-cresol sensing experiment.
HP
Radio interview confirmation.
Discuss events (escape room or workshop).
Week 7 (7/14 - 7/20)
Wet
Transform pSB1C3-FNR-native RBS-TAL, pSB1C3-FNR-B0034-TAL, pSB1C3-pchR-native RBS-GFP to E. coli top10, checked by colony PCR.
pFNR-GFP streak on plate, and put them in 37 degree incubator and anaerobic incubator.
SDS-PAGE for pSB3K3-tyrP to check protein expression.
Transformation of pSB3K3-tyrP into E. coli Nissle and E. coli BL21.
Amplify each IDT synthesis fragment.
Double digest pSB3K3-yebF-CBMBs to confirm successful of cloning.
Dry
Microfluidic chip test failed. Redesign microfluidic chip and find other ways to separate blood.
IoT connection ongoing.
Snake game programming.
Seeking possibility of CKD microbiome modelling (meeting with Prof. Liu and asking for NGS data).
HP
Arrange HP timelines.
Week 8 (7/21 - 7/27)
Wet
E. coli top10 pSB1C3-FNR-native RBS-TAL, pSB1C3-FNR-B0034-TAL, and pSB1C3-pchR-native RBS-GFP plasmid extraction and structure checking by digestion.
First attempt SDS-PAGE and western blot for E. coli top10 pSB1C3-FNR-native RBS-TAL, pSB1C3-FNR-B0034-TAL, and pSB1C3-pchR-native RBS-GFP.
SDS-PAGE for E. coli Nissle, DH5α, BL21 pSB3K3-tyrP.
PCR to add his-tag on pSB3K3-tyrP for Western Blot.
First attempt Western Blot for pSB3K3-tyrP in E. coli BL21 and Nissle.
Sequencing for pSB3K3-yebF-GS-CBMBs and pSB3K3-yebF-TB-CBMBs.
Run SDS-PAGE for bacteriocin constructs.
Dry
Team page coding.
Tweaking sensing model.
Arduino code debug.
HP
Mentor iGEM Korea_HS.
Week 9 (7/28 - 8/3)
Wet
Transform pSB1C3-FNR-native RBS-TAL, pSB1C3-FNR-B0034-TAL to E. coli Nissle, checked by colony PCR and digestion.
First functional test for pSB1C3-pchR-native RBS-GFP.
SDS-PAGE for pFNR-GFP in E. coli Nissle, samples include different time (every 8hr).
Transformation of pSB3K3-tyrP and TAL with B0034,TAL with NRBS in to same E. coli Nissle.
PCR to check if transformation of pSB3K3-tyrP and TAL with B0034 success or not.
Run SDS-PAGE and Western blot for bacteriocin constructs.
Dry
sfGFP E. coli test for detector.
Protocols & Main page in progress.
Meeting with Prof. Wu.
Populating Wiki
HP
National Education Radio Kaohsiung interview.
Week 10 (8/4 - 8/10)
Wet
Second functional test for pSB1C3-pchR-native RBS-GFP.
SDS-PAGE and western blot for E. coli top10 and E. coli Nissle pSB1C3-FNR-native RBS-TAL, pSB1C3-FNR-B0034-TAL, pSB1C3-pchR-GFP.
SDS-PAGE for pFNR-GFP in E. coli Nissle, samples include different time (4hr, 8hr, 12hr).
Dry
Start acrylic disk fabrication.
Research about promoter activity.
HP
iGEM Taiwan Conference.
Week 11 ( 8/11 - 8/17)
Wet
Second overhang extension PCR for FNR-B0034-TAL due to mutation and third overhang extension PCR for K880005-yebF-CBMB, K880005-yebF-GS-CBMBs, and K880005-yebF-TB-CBMBs.
Vector: pSB1C3 extraction.
Double digest pSB1C3, B0034-TAL, K880005-yebF-CBMB, K880005-yebF-GS-CBMBs, and K880005-yebF-TB-CBMBs. Also, ligation.
First TAL and tyrP functional assay.
SDS-PAGE for pSB3K3-tyrP.
Western Blot for TAL with NRBS/B0034.
Prepare M9 Medium for Second TAL and tyrP assay.
Dry
IPTG & Arabinose test.
Model p-cresol Growth.
HP
Go to Yunlin for volunteer with Taiwan Chronic Kidney Disease Foundation.
Geneferm entreprise visit.
NAR(National Applied Research) Lab Visit.
iGEM collaboration with iGEM UNSW.
Week 12 (8/18 - 8/24)
Wet
Transform pSB1C3- FNR-B0034-TAL, pSB1C3-K880005-yebF-CBMB, pSB1C3-K880005-yebF-GS-CBMBs, pSB1C3-K880005-yebF-TB-CBMBs to E. coli Top10. Checked by colony PCR and double digest. The right size plasmids were sent for sequencing.
Mutation found in B0034-TAL original plasmid IDT sent.
Construction of pSB1C3-pchR-B0034-GFP and transformed to E. coli DH5α.
Construction of pSB4A3-tyrP and transformed to E. coli Nissle.
pFNR-GFP fluorescent intensity test.
Dry
Experiment of p-cresol growth rate.
HP
Escape room preparation.
CKD Survey.
Education visit to Indonesia Kemurnian II High School.
Week 13 (8/25 - 8/31)
Wet
Spot on Lawn assay for pSB1C3-K880005-yebF-GS-CBMBs.
Mutation found on pSB1C3-K880005-yebF-GS-CBMBs, and pSB1C3-K880005-yebF-TB-CBMBs.
Use M9 Medium for Second TAL and tyrP assay.
Dry
Proceed acrylic disk fabrication.
HP
Write news script for NCKU News Agency.
Review Tainan First Senior High School’s feedback.
Week 14 (9/1 - 9/7)
Wet
Construction of pBBR1 MCS4-pchR-native RBS-GFP and transformation to E. coli top10.
Construction of pSB1C3-J23100-NRBS-TAL and pSB1C3-J23100-B0034-TAL to remove Fnr promoter.
Dry
Acrylic disk fabrication and test its functionality.
Test dectector’s sensitivity.
Ver3. of PDMS chip fabrication.
Model co-culture of p-cresol and clostridium
HP
Participate in NCKU Undergraduate Research.
Visit Intellectual Property Office.
Escape room preparation
Week 15 (9/8 - 9/14)
Wet
Construction of pBBR1 MCS4-pchR-native RBS-GFP and transformation into E. coli top10.
Second attempt to construct E. coli Nissle Δcan::FRT.
Construction of pSB1C3-J23100-NRBS-TAL, pSB1C3-J23100-B0034-TAL and transformation into E. coli Nissle.
Dry
Design and manufacture hardware base.
Wiki content submission.
Visit Senior of Business Administration to have a business plan and business model review.
HP
Synbiotech entreprise visit.
Week 16 (9/15 - 9/21)
Wet
Construction of pBBR1 MCS4-pchR-native RBS-GFP and transformation into E. coli top10.
Preparation of Pseudomonas fluorescens for transformation.
Test phenotype of E. coli Nissle Δcan::FRT by streaking on plates.
Construction of pSB1C3-J23100-NRBS-TAL and transformation into E. coli Nissle.
Digestion to check constructs of pSB1C3-J23100-NRBS-TAL and pSB1C3-J23100-B0034-TAL.
SDS-PAGE and Western Blot to confirm TAL and tyrP protein expression.
Dry
HP wiki page design.
Casing of device design.
Working on DFBAlab.
HP
Visit NCKU Technology Transfer & Business Incubation Center.
Visit Professor Jimmy from Business Administration to discuss about entrepreneurship.
Week 17 (9/22 - 9/28)
Wet
Construction of pBBR1 MCS4-pchR-native RBS-GFP is completed and are successful.
Preparation of Pseudomonas fluorescens for transformation.
Transformation of pBBR1 MCS-4 plasmid into P.fluorescens.
Test phenotype of E. coli Nissle Δcan::FRT by streaking on plates.
First functional test for new TAL and tyrP construct in LB Medium.
Second functional test for new TAL and tyrP construct in LB Medium with different tyrosine concentration.
Dry
Modeling E. coli Nissle pathway.
Public and Engagement wiki page design.
Finish device enclosure.
HP
Escape room.
Review business plan.
Week 18 (9/29 - 10/5)
Wet
Test phenotype of E. coli Nissle Δcan::FRT by streaking on plates.
Preparation of Pseudomonas aeruginosa for transformation.
Test antibiotic resistance of Pseudomonas fluorescens 55.
Dry
Finish all wiki page design.
Finish SolidWorks illustration.
Finish E. coli Nissle & Clostridium difficile metabolic network.
Filming demonstration video.
HP
Collaboration with Washington iGEM.
Visit Winston Medical Corporation.
Finalized business plan.