Team:NYCU-Taipei/Progress

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Check out what we have done in these months!
June
Wet Lab
  • Week 4
    All team members received online lab safety lectures.

Human Practices
In June, we
  • Reached out to Mr. Katsuhito Hayashi from the University of Tokyo, and with his help, got in contact with “Dr. Natto, ” Prof. Hiroyuki Sumi, who discovered Nattokinase, and Prof. Chieko Yatagai from Kurashiki University of Science and The Arts.

  • Reached out to Dr. Shih-Lin Chang and held an online interview; were suggested to narrow down the claimed target of our thrombosis detection kit to deep vein thrombosis (DVT), and were inspired to do a elders community education on DVT.
July
Wet Lab
  • Week 2
    Three wet lab leaders received lab training with our PIs and NYMU-Taipei 2020 team members. Also we got our kind gift “ B. subtilis  HS01 & HS46 “ from National Kaohsiung University of Science and Technology.
  • Week 3
    Four other team members received lab training with our PIs and lab leaders. Besides, we successfully extracted gDNA from E. coli  MG1655 and B. subtilis   HS01 & HS46.
  • Week 4
    All of our team members finished lab training. We did a comprehensive clean up of our lab, including sterilizing containers, throwing away expired solutions and putting on new bench paper.

Human Practices
In July, we
  • Decided to use Nattokinase as our thrombolytic enzyme after exchange of ideas with Prof. Sumi and Prof. Yatagai.
  • Reached out to Prof. Ying-Chieh Tsai and held an online interview; were suggested to not use any antibiotics with the removal of E. coli , and were introduced with the concept of “Live Biotherapeutic Product (LBP)”.
  • Consulted advice from Dr. Jaw-Wen Chen, and were reminded of the importance of designing a way to thoroughly remove our engineered E. coli  when needed, due to concerns of special circumstances such as having an operation.
  • Decided to implement a kill switch design into our project, after continual exchange of ideas with Mr. Katsuhito, for minimum disturbance to the gut microbiota.
  • Reached out to the chief of Yi-Ching Yuan Elderly Long-Term Care Center, Mr. Chih-Sung Yu, and gathered more insight on the importance of preventive medicine for the elders.
  • Participated in the iGEM Taiwan Meetup.
August
Wet Lab
  • Week 1
    We amplified iGEM kits (i.e. J32015, B0012, I15008, K763004, etc.) for further use in different parts of our design.
  • Week 2
    We amplified pET-21a cloning vectors for Nattokinase production and Optogenetic system. Also, we kept on amplifying iGEM kits (i.e. B0034,J23106) for further use.
  • Week 3
    We cloned aprN  gene from B. subtilis  HS01 & HS46. Besides, we failed in ligating our kill-switch parts due to misuse of T4 ligase buffer. For our optogenetic parts, we ligated BBa_I15008 and BBa_B0015, and successfully transformed the ligation product(1) into E. coli  DH5-alpha.
  • Week 4
    We set up and revised PCR programs for cloning aprN gene. Besides, for parts in our optogenetic system, we amplified the transformed plasmid(1) and confirmed the ligation was successful. Then, we ligated it with BBa_K763004, and successfully transformed the ligation product(2) into E. coli  DH5-alpha.
  • Week 5
    We successfully cloned aprN  gene without signal peptide sequence from B. subtilis  HS01 & HS46. For our optogenetic system, we amplified the transformed plasmid(2) and confirmed the ligation was successful. Also, plasmid number pKA207I10 that we ordered from Addgene arrived as well as the primer we ordered for BphP1, Q-PAS1 , and LexA  too. Lastly, we started our first round freeze-drying of E. coli  K12 MG1655.

Human Practices
In August, we
  • Decided to integrate the concept of LBP into our project design after gaining more understanding of it, under continual communication with Prof. Tsai through emails and phone calls.
  • Held a physical interview with Dr. Chen, and was encouraged to consider linking primary prevention or secondary prevention to our project.
  • Decided to integrate the concept of preventive medicine into our project.
  • Reached out to Biorich Bio Technology Co. Ltd., and consulted their advice on the selection of freeze-dry techniques.
  • Reached out to MegaPro Biomedical Co., Ltd. for advice on our oral delivery system design, and was suggested to not implement too many new designs on one single project to simplify the attribution of different designs in our project.
  • Reached out to Prof. Padma Devarajan for advice on our oral delivery system design, and was suggested to simplify our design.
  • Sent out a public survey to our community and also to the iGEM collaboration page to gather opinions on our project, including the oral delivery system.
  • Decided to abandon our original oral delivery system design, and reached out to Dah Feng Capsule Industry Co., Ltd. (DFC) to try to get a sponsor.
  • Posted information of the “2021 iGEM International Optogenetics Conference” to be held in September on the iGEM collaboration page, and sent invitation letters to iGEM teams with optogenetic designs this year.
  • Got in touch with iGEM team TAS_Taipei, expressed interest in doing a collaboration with them.
  • Starting to post on social media platforms with information on synthetic biology, iGEM, our team project and progress, and information for thrombosis.
  • Got in contact with the “Taipei First Girls High School Biology Club (TFGBC)” and agreed upon doing an education session in September.
September
Wet Lab
  • Week 1
    For the kill switch, we ligated products from the past two weeks to form functional units and successfully cloned the MazE/MazF  gene. Also, we extracted gDNA from E. coli  BL21 DE3 to use as a PCR template for adhesion protein. As for our optogenetic parts, we successfully cloned Bphp1  and QPAS1  from template pKA207I10; however, we discovered that the restriction enzyme cutting site that we added on the downstream of BphP1 cloning product was also contained in the sequence of BphP1. So, we re-designed the primer for BphP1  cloning.
  • Week 2
    We constructed our plasmid-NK by ligating aprN  to pET-21a. For adhesion protein, we transformed the iGEM part “BBa_E0040” into E. coli  DH5-alpha and cloned the FimH  and OmpA  gene from BL21 to construct our parts. For our optogenetic parts,we failed to clone LexA from the genome of E. coli  MG1655 several times. We suggested that the problem may originate from the primer solution. Thus, we re-prepared the primer solution for LexA  cloning and it worked out. Besides, we inserted QPAS1  and LexA  into pET-21a respectively and successfully transformed the ligation product into E. coli  DH5-alpha and confirmed both were successful. Lastly, we freeze-dried E. coli  DH5-alpha and ran preliminary data analysis.
  • Week 3
    We failed to construct plasmid-proNK with signal peptide because the colonies we picked were wrong. Besides, we started preparing kill-switch parts for Gibson assembly. For optogenetic parts, we successfully cloned PCR products for Gibson assembly containing BphP1, QPAS1  and LexA  genes. Also, we inserted BBa_B0015 into pET-21a and transformed the product into E. coli  DH5-alpha. The ligation was confirmed successful. Lastly, we cloned the GFP  gene from iGEM part “BBa_E0040" for our design in adhesion protein .
  • Week 4
    This week, we got very low concentration of results after every gel extraction. We found out that some subpackage of Elution buffers had been contaminated. For NK production, we transformed our plasmid-NK into BL21 successfully. Also, we made fibrin plate without filter paper, resulting in bad formulation of fibrin clot. For the kill switch, we amplified iGEM kits (i.e. J23112, J23113, J23109, J23116, J23105, J23110, J23118, J23102) to replace our previous promoter in order to improve our system regulation. For optogenetic parts, the oligonucleotide named PR that we ordered arrived. We inserted the PR fragment into BBa_B0015 after amplifying the PR fragment and successfully transformed the ligation product(3) into E. coli  DH5-alpha. Lastly, we freeze-dried E. coli  DH5-alpha for more data groups.

Dry Lab
In September, we
  • Determined the direction of our project. Since the cleavage sites of nattokinase remain unelucidated, we are trying to use a software model to make the prediction.
  • Surveyed some related works for our software project. We referred to several pieces of research for our model and algorithm design.
  • Found some open source tools for our implementation.
  • Crawled all the data we needed in the project and also preprocessed them for our uses.

Human Practices
In September, we
  • Held the “2021 iGEM International Optogenetics Conference”, exchanged project design ideas with iGEM team KUAS_Korea.
  • Visited the headquarter of DFC in Taichung, and were sponsored with their acid resisting capsule.
  • Designed the health education leaflet on DVT, and verified the information on it with Dr. Chang.
  • Visited Yi-Ching Yuan Elderly Long-Term Care Center, and gave out health education leaflets on DVT.
  • Reached an agreement with iGEM team TAS_Taipei on doing a collaboration on experimental verification.
  • Held an educational session with TFGBC.
October
Wet Lab
  • Week 1
    Firstly, we checked plasmid extracted from BL21 to know whether transformation of plasmid-NK was successful. For the kill switch, from the modeling data we got, we realized our TetR promoter is hard to regulate, resulting in unexpected wet lab results. For optogenetic parts, we cloned the PCR product for Gibson assembly containing product(3). There are two different products. One is for BphP1  expression, and the other is for LexA_QPAS1  fusion protein expression. For adhesion protein, we ordered a new primer pair for our construct due to incorrect design( double cleavage site ) after routine failure in enzyme digestion of OmpA, FimH, GFP  and pET-21a with wrong band numbers.
  • Week 2
    We revised our way of making fibrin plate by adding a filter paper on top, resulting in uniform fibrin clot formation. Then, we established the urokinase standard curve. For our detection device, we printed out our gadget by using a 3D-printer and established our D-dimer standard curve. For our optogenetic parts, we inserted fragment (2), which is from August Week 5, into pET-21a. Also, the primer designed for expressing BphP1, QPAS1,  constructing fusion protein BphP1_mCherry, QPAS1_mCherry, LexA_QPAS1,  and LexA_QPAS1_mCherry  arrived. Lastly, we made E. coli  Nissle 1917 competent cells and freeze-dried for our oral delivery system.
  • Week 3
    Firstly, we expressed and purified Nattokinase from BL21. However, no protein was produced due to the wrong induction temperature. As for adhesion protein, we successfully constructed FimH, OmpA,  and GFP in pET-21a. For the optogenetic system, we successfully cloned PCR products after getting our primer last week. Then, we inserted BphP1, mCherry,  and QPAS1  into pET-21a and successfully transformed it into E. coli  DH5-alpha. The insertion was confirmed successful after amplifying the transformed plasmid. Besides, we assembled all the fragments into the vector without part (2) and successfully transformed the Gibson assembly product into E. coli  DH5-alpha. Lastly, we constructed our second design of kill switch with Tendon promotor after the conclusion we got from Oct Week 1.
  • Week 4
    We expressed and purified Nattokinase from BL21 again with modified temperature. Then, we ran SDS-PAGE to confirm the size of protein; fibrin plate and spectrophotometer were used to see the activity of our Nattokinase. For optogenetic parts, we inserted QPAS1  into plasmid containing mCherry on the backbone of pET-21a. For adhesion protein, we induced and purified FimH  in BL21 and ran SDS-PAGE to confirm our FimH  protein.
Dry Lab
In October, we
  • Confirmed the framework and formulation of our project.
  • Implemented our model and algorithm.

Human Practices
In October, we
  • Conducted the collaboration experiments with team TAS_Taipei.
  • Contacted every expert and iGEMers who contributed to our project for their agreement on us putting their information on our special thanks page.
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