Team:NCTU Formosa/Team

From 2012.igem.org

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Revision as of 07:56, 26 September 2012

Team:NCTU Formosa - 2012.igem.org

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　 Chang, Ting

Hello everyone! I am Zoe. I love to swim and drawing pictures.I am happy to join in this iGEM group. Many things I have learned from the process. It is truely a good experience to me forever. Zoe is the head of experiment group 1 which are responsible for assembling the first half circuits of our i-butanol-producing enzymes. She is the chief designer of our animation.

　 Hsieh, Hsin-Yun

Hello, She's Lisa Hsieh. She enjoys the process of the experiment with teammates. She likes to draw pictures. With a little perfectionism, it took her 2 days to finish team's mascot. She especially likes to drink a cup of hot cocoa after a long day work, and she believes cocoa can cure all diseases! Lisa worked with Zoe and Pearl to construct PSB II + ilvC and Ptet + B0032 + Gli1. She is the designer of our cute robot mascot.

　 Yu, Chun-Wei

Chun-Wei is a 3rd year biotechnology student at NCTU, love nature and playing guitar also love biology. He joined iGEM to get involved in the world-changing field of synthetic biology. Together with Jenny, Chun-Wei assembled the first two part of our enzyme series, and did the tolerance experiment. He is also an important role in our Human practice who trained students to be our human practice staffs.

　 Chen, Wei-Ting

Vicky Chen is a junior in National Chaio Tung University, major in bio-technology. She likes spending time in wet lab, discussing with her teammates. She enjoys eating foods ,shopping and traveling. Vicky assembled zif268 and alsS and optimized the growth condition in which E. coli can produce most i-butanol with Yu-Che.

　 Wu, Cheng-Hsuan

Wu, CHENG-HSUAN (English name: Max) is a third year Biological Science and Technology at NCTU. He enjoys playing basketball and going around the night markets. You can contact CHENG-HSUAN at max810327@hotmail.com.tw Max is the head of experiment group 2 which are responsible for assembling the second half circuits of our i-butanol-producing enzymes. Our instrument is mainly designed by him.

　 Lee, Chia-Jung

As a student majoring in biological science and technology in the third year in NCTU, Chia-Jung always want to know the logic and consequences behind things around him. He likes Chemistry and Biology particularly. In his free time, he likes playing tennis and training to improve his skill. Chia-Jung is the leader of 2012 NCTU-Formosa. He worked with group 2 to assemble biobrick and participated in summer camp as a TA.

　 Yu, Chieh-Cheng

Charlie white yu is a third-year student National Chiao Tung University who is majoring in Biological Science and Technology. He thought iGEM would be a great opportunity to further his knowledge of genetics, gain valuable research experience, and explore possible area of interest. Charlie is in charge of survey jobs and souvenir design. He was a menber of group 2 and also a TA in our summer camp.

　 Lin, I

Hi, my name is Lin I. I am from Taiwan and I major in Biotechnology in National Chiao Tung University. Playing basketball and listening classical music are my hobbies. It is really exciting and pleasant that I can be one of the 2012 iGEM team members. However, this is not all about me. If you have willing to contact me, maybe we will become friend! I is the “pro” of constructing biobricks in group 2. No matter how hard is it, I can built it. He also made our opening clip with Kyle.

　 Peng, Kai-Chun

Hi! I'm Kyle, a third year student contributing in Bio-technology at National Chiao Tung University of Taiwan. I'm interest in planting a variety of carnivorous plants which are so beautiful and enigmatic. I’m looking forward to meeting other school team. I hope it may be a real eye-opener for me. Besides being a member of group 2, Kyle played an important role in our summer camp. He edited handout written by TAs, designed courses, experiments and iGEM lectures. And he is the head of our video team which made our opening clip and animation.

　 Tsai, Lu-An

TSAI, Lu-An is a Qusai-Bachelor of NCTU, the adherent of Adeptus scientia and Preacher of capitalism. He loves the smell of napalm in the morning, the view of chaos in parliament. By using strange logic and weird metaphor, he can confuse the listeners, and Unfortunately himself. Besides being a member of group 2, Lu-An is the head of wiki group which made our beautiful team wiki. He also gave a lecture to students with pearl in summer camp.

　 Chung, Chia-Jung

Hello, I'm Pearl. I like to do experiments, and I go to lab rain or shine even on typhoon. Besides that, I like to chat with friend. I wear a big smile on my face .I like to practice cheerleading, too. Together with Lisa and Zoe, Pearl to construct PSB II + ilvC and Ptet + B0032 + Gli1. And she is the main designer of our T-shirt. She also gave a lecture to students with Lu-An in summer camp.

　 Cheng, Yu-Che

CHENG Yu-Che is a third year biotechnology student at National Chiao-Tung University. He's addicted to synthetic biology and molecular biology. So he may go this way in the future and keep playing table tennis which he is good at. Yu-Che assembled zif268 and alsS and optimized the growth condition in which E. coli can produce most i-butanol with vicky.

　 Soo, Ching-Ren

Soo, Ching-Ren is a Biological Science and Technology at NCTU. He is from Malaysia, hobby is play video games. He enjoys experiment of IGEM, he think it will very usefull in his future. Ching-Ren worked with Lisa Pearl and Zoe to assembled Ptet + B0032 + Gli1 + kivd + B0015

　 Hsu, Tzu-Yun

Jenny Hsu is a third-year student who is majoring in Biotechnology in National Chiao Tung University. She enjoys doing the experiments, cooking, and making handicraft. She thinks that attending 2012 iGEM is a good experience to her, and hopes she can make many friends in iGEM and share knowledge with each other. Jenny assembled the first two part of our enzyme series, and did the tolerance experiment with Chun-Wei. She also played an important role in summer camp. She connected our department for fund and gathered students to make our plan come true.

　 Calvin Hue

I’m a Photonics major at NCTU. I was overwhelmed with fun and experienced the power of Synaptic Biology during our cooperation. It’s also my pleasure to get involved in this project and make new friends. At the moment, I'm mainly working on the wiki site. I'm trying to make it eye-pleasing and user-friendly.

　 Tseng, Ching-Ping

He is a Professor of Biological Science and Technology at National Chiao Tung University, Hsinchu, Taiwan, R.O.C. Highest Education: Ph. D. Rutgers University, USA, 1991.Since 2001, Tseng has been a professor in NCTU, Taiwan. His current research interests include molecular biology, synthetic biology, applied microbiology, and bioengineering.

　 Lee, Hsiao-Ching

Received the B.S. degree and Ph.D. degree from the Department of Life Science at National Tsing Hua University, Hsinchu, Taiwan, R.O.C. in 1999 and 2005 respectively. From 2005 to 2007, she worked as a postdoctoral research position at NTHU. She is now an Assistant Professor with the Department of Biological Science and Technology at National Chiao Tung Universit. Her current research interests include molecular biology, synthetic biology and bioinformatics.

　 Huang, Hsien-Da

Dr. Huang is a Professor and the current Chairman of Department of Biological Science & Technology, Institute of Bioinformatics and Systems Biology of National Chiao Tung University, Hsinchu, Taiwan R.O.C. His current research include microRNA regulation and protein post-translational modification databases building and dry-lab tools development.

　 Yu-Pei Chen

Received the Ph.D. degree from the Department of Biological Science and Technology at National Chiao Tung University, Hsinchu, Taiwan, R.O.C. in 2008. From 2008 to 2012, he worked as a postdoctoral research position in Department of Biological Science and Technology and Mechanical Engineering at NCTU. His current research interests include molecular biology, biochemistry and fungi secondary metabolite.

　 Wu, Shao-I

I am a master student, majoring molecular medicine and bioengineering. Hobby: travel, photography, music, volleyball.

　 Tseng, De-Jung

I’m De-Jung Tseng, a fourth grade student of National Chiao Tung University. Talented at fine art and literary creation , I have high curiosity of everything in daily life and plenty of ideas. Believing in the innovation of science ,I'm passionate to have fun in iGEM 2012. The limit is the mark which would be overcome next. I designed our beautiful souvenir.Wish you all would like it.

　 Chang, Shu-Han

As a student majoring in biological science and technology of National Chiao-Tung University, I have the chance to join the competition. Although we have lots of problem to solve, but we never give up and finish the job. Since then, I love my partners so much.

　 Malvin Jefri

An International student in NCTU, majoring in Biological Science and Technology. I'm a badminton freak who loves watching Korean serial movies and Korean boy bands. It's my pleasure to join IGEM competition to taste what's so called teamwork in doing experiment as a team. There's so much sweet n sour moment during these period. Last, I'm just An ordinary Indonesian boy who does ordinary stuffs but always planned to go beyond my limit.

Host Lab

In this year preparation, most of our experiments were done in the Applied Microbiology & Biochemistry Lab, which also known as CPT Lab named after supervisor prof. Ching-Ping Tseng. CPT Lab locates at National Chiao Tung University (Boai campus), Hsinchu, Taiwan, R.O.C.

　

Come visit the website of our lab: http://life.nctu.edu.tw/~cptlab/Home.html

Gallery

Notes

March 2012

Sunday

Monday

Tuesday

Wednesday

Thursday

Friday

Saturday

11

12

13

  • mini-prep of cultivated AlsS, pSB1K3, pSB1A3, and pSB1C3 E.coli (G2)

14

  • digestion : [Zif268] ES & [AlsS] XP & [pSB1C3] EP (G2)

  • ligation : insert[Zif268] ES & [AlsS] XP/Vector[pSB1C3] EP (G2)

15

16

  • Transformation of B0034

  • Cultivation of transformed E.coli on LBA plate

17

18

19

  • Transformation of Zif268+AlsS+pSB1C3 in DH5-alpha (G2)

  • Point mutation HivC (PCR, DPN1 digest, TA cloning, transform) (G2)

20

  • PCR of point mutation HivC (G2)

  • PCR of insert fragment [Zif268+AlsS] (G2)

21

  • electrophoresis of insert fragment [Zif268+AlsS]----NOT OK (G2)

22

  • Single colony isolation from pLac LBA plate, and cultivation of pLac E.coli in liquid LBA

  • Single colony isolation from pSB1K3 LBK plate, and cultivation of pSB1K3 E.coli in liquid LBK

23

  • mini-prep of cultivated pLac & pSB1K3 E.coli

24

  • digestion : [pSB1K3] EP

25

26

27

28

29

  • Single colony isolation from Zif268 LBA plate, and cultivation of Zif268 E.coli in liquid LBA

  • Single colony isolation from AlsS LBA plate, and cultivation of AlsS E.coli in liquid LBA

30

  • mini-prep of cultivated Zif268 Ecoli & AlsS Ecoli

  • digestion :[Zif268] ES/[AlsS] XP

31

April 2012

Sunday

Monday

Tuesday

Wednesday

Thursday

Friday

Saturday

1

  • ligation : insert Zif268 [ES] & AlsS [XP]/Vector pSB1K3 [EP]

2

  • transformation of Zif268+AlsS+pSB1K3 and cultivation on LB-K plate

3

4

  • digestion : [pSB1K3] EP

  • digestion : [pSB1K3] EP

5

6

7

  • PCR of insert fragment [Zif268+AlsS] OK

  • DNA Sequencing OK

  • ligation : point mutation HivC

  • TA cloning : point mutation HivC

8

9

  • Single colony isolation from Zif268+AlsS LB-K plate, and cultivation of in liquid LB-K

10

  • mini-prep of cultivated Zif268+ AlsS E. coli

  • transformation of point mutation HivC and cultivation on LB-A plate

  • transformation of B0034 and cultivation on LB-A plate

11

12

  • Single colony isolation from HivC LB-A plate, and in liquid LB-A

  • Single colony isolation from B0034 LB-A plate, and in liquid LB-A

13

  • mini-prep of cultivated point mutation HivC E. coli & B0034 E. coli

14

  • digestion : Zif268+AlsS [XP]

15

16

17

  • Single colony isolation from ilvD LB-A plate, and cultivation in liquid LB-A

18

  • mini-prep of cultivated ilvD E. coli

  • digestion : ilvD [EP] & pSB1K3 [EP]

  • transformation of 37℃ RBS and cultivation on LB-C plate

19

  • Single colony isolation from HivC LB-A plate, and cultivation in liquid LB-A

20

  • digestion : B0034 [SP]

  • gel extraction

  • ligation :insert Zif268+AlsS [XP]/Vector B0034 [SP]

  • mini-prep of cultivated Hivc E. coli

21

  • transformation of B0034+Zif268+AlsS and cultivation on LB-A plate

  • transformation of HivC and cultivation on LB-A plate

22

  • PCR of insert fragment [B0034+Zif268+AlsS] OK

  • DNA Sequencing NOT OK

  • digestion : B0034 [SP] & Zif268+AlsS [XP]

  • gel extraction

  • ligation :insert Zif268+AlsS [XP]/Vector B0034+pSB1A3 [SP]

  • transformation of B0034+Zif268+AlsS and cultivation on LB-A plate

  • Single colony isolation from B0034 LB-A plate, and cultivation in liquid LB-A

  • SCI from ilvD LB-K plate, and cultivation in liquid LB-K

23

  • PCR of insert fragment [B0034+Zif268+AlsS] NOT OK

  • transformation of B0034+Zif268+AlsS and cultivation on LB-A plate

  • mini-prep of cultivated B0034 and ilvD E. coli

  • Single colony isolation from HivC LB-A plate, and cultivation in liquid LB-A

24

  • mini-prep of cultivated HivC E. coli

  • digestion : B0034 [SP] & ilvD [XP]

25

  • digestion : pSB1C3 [EP] & HivC [ES] & HivC [SP]

  • ligation :insert HivC [ES] & ilvD [XP]/Vector pSB1C3 [EP]

  • ligation :insert ilvD [XP]/Vector HivC+pSB1A3 [SP]

26

  • transformation of HivC+ilvD and cultivation on LB-A & LB-C plate

27

  • PCR of insert fragment [B0034+Zif268+AlsS] NOT OK

28

  • transformation of pSB1A3 & pSB1C3 and cultivation on LB-A & LB-C plate

  • PCR of insert fragment [HivC+ilvD] OK

  • DNA sequencing NOT OK

29

  • Single colony isolation from pSB1C3 LB-C plate, and cultivation in liquid LB-C

30

  • mini-prep of cultivated & pSB1C3 E. coli

May 2012

Sunday

Monday

Tuesday

Wednesday

Thursday

Friday

Saturday

1

  • digestion : pLac [EP] & pSB1K3 [EP]

  • ligation :insert pLac [EP]/Vector pSB1K3 [EP]

  • transformation of pLac+pSB1K3 and cultivation on LB-K plate

2

  • PCR of insert fragment [pLac+pSB1K3] OK

  • DNA Sequencing OK

  • transformation of B0034_new and cultivation on LB-A plate

  • Single colony isolation from B0034 LB-A plate, and cultivation in liquid LB-A

3

  • mini-prep of cultivated B0034 E. coli

  • digestion : B0034 [SP]

  • ligation :insert Zif268+AlsS [XP]/Vector B0034+pSB1A3 [SP]

  • transformation of B0034+Zif268+AlsS

  • cultivation on LB-A plate

4

  • PCR of insert fragment [B0034+Zif268+AlsS]-----self ligation

  • transformation of 37℃ RBS and cultivation on LB-C plate

5

6

7

8

  • transformation of 37℃ RBS and cultivation on LB-Kplate

9

  • Single colony isolation from 37℃ RBS LB-K plate, and cultivation in liquid LB-K

10

  • digestion : B0034 [ES] & pSB1C3 [EP]

  • mini-prep of cultivated 37℃ RBS E. coli

11

  • ligation :insert B0034 [ES] & pSB1C3 [EP]/Vector pSB1C3 [EP]

  • transformation of B0034+Zif268+AlsS and cultivation on LB-C plate

12

  • digestion : pLac [ES] & pLac [SP]

  • digestion : B0034 [SP] Kr (gel extraction) , B0034 [SP] Ar (gel extraction) , 37℃ RBS [XP] & HivC [XP]

  • ligation : (1. 2 parts) insert HivC [XP], Vector B0034+pSB1K3 [SP]

  • ligation : (2. 3 parts) insert HivC [XP], Vector B0034+pSB1A3 [SP]

13

14

15

  • PCR of insert fragment [B0034+Zif268+AlsS] OK

  • DNA Sequencing OK

  • transformation of B0034+ HivC and cultivation on LB-A & LB-K plate

16

17

18

  • Single colony isolation from pSB1A3 LB-A plate, and cultivation in liquid LB-A

  • Single colony isolation from B0034+Zif268+AlsS LB-C plate, and cultivation in liquid LB-C

  • Single colony isolation from B0034+HivC LB-A & K plate, and cultivation in liquid LB-A & K

19

  • mini-prep of cultivated pSB1A3 E. coli & B0034+Zif268+AlsS

  • digestion: pSB1A3 [EP] & B0034+Zif268+AlsS [XP]

  • ligation: (1.3 parts)insert pLac [ES] & B0034+Zif268+AlsS [XP], Vector pSB1A3 [EP]

  • ligation: (2.2 parts)insert B0034+Zif268+AlsS [XP], Vector pLac+pSB1K3 [SP]

  • transformation of pLac+B0034+Zif268+AlsS and cultivation on LB-A plate & LB-K plate

  • mini-prep of cultivated B0034+HivC (Ar & Kr)

20

  • PCR of insert fragment [pLac+B0034+Zif268+AlsS] OK

  • DNA Sequencing 3 parts OK

21

  • digestion : 37℃ RBS [XP] , ilvD [ES] & pSB1C3 [EP]

22

23

  • digestion : HivC+ilvD-3,18 [EP]

  • ligation :insert ilvD[ES] & 37℃ RBS [XP], Vector pSB1C3 [EP]

24

  • Single colony isolation from pLac+B0034+Zif268+AlsS LB-A plate, and cultivation in liquid LB-A

25

  • mini-prep of cultivated pLac+B0034+Zif268+AlsS E. coli

  • transformation of ilvD+37℃ RBS and cultivation on LB-C plate

26

  • PCR of insert fragment [ilvD+37℃]-----OK

  • DNA Sequencing OK

27

28

29

30

  • Single colony isolation from PBSII+ilvC LB-K plate, and cultivation in liquid LB-K

  • ligation : insert HivC [XP], vector B0034+pSB1A3 [SP]

31

  • mini-prep of cultivated PBSII+ilvC E. coli

  • digestion : pSB1C3 [EP] & PBSII+ilvC [XP]

  • ligation : insert B0034 [ES] & PBSII+ilvC [XP], vector pSB1C3 [EP]

  • transformation of B0034+PBSII+ilvC and cultivation on LB-C plate

  • transformation of B0034+HivC and cultivation on LB-A plate

June 2012

Sunday

Monday

Tuesday

Wednesday

Thursday

Friday

Saturday

1

  • PCR of insert fragment [B0034+PBSII+ilvC] OK!

  • DNA Sequencing OK!

2

3

  • mini-prep of cultivated B0034+PBSII+ilvC E. coli & pLac+B0034+Zif268+AlsS E. coli

  • mini-prep of cultivated HivC+ilvD-12,20 E. coli

    • 4

    • digestion : HivC+ilvD-12,20 [XP]

    5

    • Design the primer for pLac+B0034+Zif268+AlsS point mutation (pm)

    6

    • digestion : B0034 [ES] & HivC [XP] & pSB1C3 [EP] & HivC [EP]

    • ligation : insert B0034 [ES] & HivC [XP]/vector pSB1C3 [EP]

    • ligation : insert HivC [EP]/vector pSB1C3 [EP]

    • transformation of B0034+ HivC & HivC ,and cultivation on LB-C plate

    7

    • PCR of insert fragment [B0034+ HivC] & [HivC] OK

    8

    • Design the primer for B0034+PBSII+ilvC point mutation (pm)

    • Single colony isolation from HivC, B0034+HivC & ilvD+37℃ RBS LB-C plate, and cultivation in liquid LB-C

    9

    • mini-prep of cultivated HivC & B0034+HivC & ilvD+37℃ RBS E. coli

    • Testing the temperature of the point mutation between of HivC & ilvD by the m.p 50℃ of PCR

    • ligation: insert B0034+HivC [ES] & ilvD [XP]/vector pSB1A3 [EP];insert B0034+HivC [ES] & ilvD+37℃ RBS [XP]/vector pSB1A3 [EP]

    10

    • transformation of B0034+ HivC+ ilvD & B0034+ HivC+ ilvD+37℃ RBS ,and cultivation on LB-A plate

    • Testing the temperature of the point mutation between of pLac+B0034+Zif268 & AlsS by the m.p 55℃ of PCR failed

    11

    • DNA Sequencing B0034+HivC & HivC OK

    • PCR of insert fragment [B0034+ HivC+ilvD] & [B0034+ HivC+ilvD+37℃ RBS] OK

    12

    • Single colony isolation from B0034+ HivC+ilvD+37℃ RBS & B0034+ HivC+ ilvD LB-A plate, and cultivation in liquid LB-A

    • Single colony isolation from HivC(TA) LB-A plate, and cultivation in liquid LB-A

    13

    • mini-prep of cultivated B0034+ HivC+ilvD+37℃ RBS , B0034+ HivC+ ilvD & HivC(TA) E. coli

    • Testing the temperature of the point mutation between of pLac+B0034+Zif268 & AlsS by the m.p 50 & 52℃ of PCR----50℃ is better

    14

    • PCR of insert fragment [pLac+B0034+Zif268+AlsS] with designed primer for point mutation -----NOT OK

    15

    • DNA Sequencing B0034+ HivC+ilvD+37℃ RBS & B0034+ HivC+ ilvD OK

    16

    17

    18

    19

    • PCR of insert fragment [pLac+B0034+Zif268+AlsS] with designed primer for point mutation OK

    • digestion: pLac+B0034+Zif268+AlsS [DPn1]

    20

    • transformation of pLac+B0034+Zif268+AlsS (point mutation), and cultivation on LB-A plate

    21

    • Single colony isolation from pLac+B0034+Zif268+AlsS (pm) LB-A plate, and cultivation in liquid LB-A

    22

    • mini-prep of cultivated pLac+B0034+Zif268+AlsS (pm) E. coli

    • digestion : pLac+B0034+Zif268+AlsS (pm) [EP]

    23

    • DNA sequencing OK

    24

    25

    • PCR of insert fragment [B0034+ PBSII+ilvC] with designed primer for point mutation OK

    • digestion : B0034+ PBSII+ilvC [DPn1]

    26

    • transformation of B0034+ PBSII+ilvC (pm), and cultivation on LB-C plate------Fail

    27

    • PCR of insert fragment [B0034+ PBSII+ilvC] with designed primer for point mutation OK

    28

    • digestion : B0034+ PBSII+ilvC [DPn1]

    29

    • transformation of B0034+ PBSII+ilvC (pm), and cultivation on LB-C plate

    30

    • PCR of insert fragment [B0034+ PBSII+ilvC] (pm) OK

    • Single colony isolation from B0034+ PBSII+ilvC (pm) LB-C plate, and cultivation in liquid LB-C

    July 2012

    Sunday

    Monday

    Tuesday

    Wednesday

    Thursday

    Friday

    Saturday

    1

    • mini-prep of cultivated B0034+ PBSII+ilvC (pm) E. coli

    • digestion : B0034+ PBSII+ilvC (pm) [EP]

    2

    • DNA sequencing OK

    3

    4

    5

    6

    7

    • digestion : pLac+B0034+zif268+AlsS (pm) [ES] & B0034+ PBSII+ilvC (pm) [XP]

    8

    9

    • Testing the temperature of the point mutation between of HivC & ilvD by the m.p 50℃ of PCR

    • digestion : pSB1K3 [EP]

    • ligation : insert pLac+B0034+zif268+AlsS (pm) [ES] & B0034+ PBSII+ilvC (pm) [XP]/vector pSB1K3 [EP]

    • transformation of pLac+B0034+zif268+AlsS+ B0034+ PBSII+ilvC (pm) and cultivation on LB-K plate

    10

    • Testing the temperature of the point mutation between of HivC & ilvD by the m.p 48℃ of PCR

    • Digestion : B0034+ HivC+ilvD+37℃ RBS [DPn1]

    • PCR of insert fragment [pLac+B0034+zif268+AlsS+ B0034+ PBSII+ilvC] (pm) OK

    • SCI from pLac+B0034+zif268+AlsS+ B0034+ PBSII+ilvC (pm) LB-K plate, and cultivation in liquid LB-K

    11

    • mini-prep of cultivated pLac+B0034+zif268+AlsS+ B0034+ PBSII+ilvC (pm) E. coli

    • DNA sequencing OK

    • culture condition test: activation DH5αovernight

    12

    • transfer to new medium(1/100), OD0.2 start counting culture time

    • transfer to 30゜C and 27゜C

    13

    • transfer to 30゜C and 27゜C

    14

    15

    • transformation of B0034+ HivC+ ilvD+37℃ RBS (pm),and cultivation on LB-A plate

    16

    • PCR of insert fragment [B0034+ HivC+ilvD+37℃ RBS](pm)-----NOT OK

    17

    • Point mutation by PCR of insert fragment [B0034+ HivC+ilvD+37℃ RBS]

    • Digestion: B0034+ HivC+ilvD+37℃ RBS [DPn1]

    18

    19

    • transformation of B0034+ HivC+ ilvD+37℃ RBS (pm),and cultivation on LB-A,K,C plate (A sucessful)

    20

    • PCR of insert fragment [B0034+ HivC+ilvD+37℃ RBS](pm) ---- OK

    • Single colony isolation from B0034+ HivC+ilvD+37℃ RBS (pm) LB-A plate, and cultivation in liquid LB-A

    21

    • mini-prep of cultivated B0034+ HivC+ilvD+37℃ RBS (pm) E. coli

    • Digestion: pLac+B0034+zif268+AlsS+ B0034+ PBSII+ilvC (pm)(G1) [ES] & B0034+ HivC+ilvD+37℃ RBS (pm)(G2) [XP]

    • ligation : insert G1[ES] & G2[XP] vector pSB1C3[EP]

    22

    • transformation of G1+G2,and cultivation on LB- C plate failed

    23

    • ligation : insert G1[ES] & G2[XP]/vector pSB1C3[EP]

    24

    • transformation of G1+G2,and cultivation on LB- C plate

    • Digestion: G2’(B0034+HIVC+ilvD) [XP]

    • ligation : insert G1[ES] & G2‘[XP]/vector pSB1C3[EP]

    25

    • PCR of insert fragment [kivD+B0015]

    • transformation of G1+G2’, and cultivation on LB- C plate failed

    26

    • Single colony isolation from kivD+B0015 LB-C plate, and cultivation in liquid LB-C

    • sample and do GC

    27

    • mini-prep of cultivated kivD+B0015 E. coli

    28

    29

    • Point mutation by PCR of insert fragment [B0034+ HivC+ilvD(G2’)]

    • Digestion: G2’ [DPn1]

    30

    • transformation of G2’, and cultivation on LB- A plate

    31

    • Digestion: kivD+B0015 [EP] (checking bp----failed)

    • Single colony isolation from G2’ (pm) LB-A plate, and cultivation in liquid LB-A

    August 2012

    Sunday

    Monday

    Tuesday

    Wednesday

    Thursday

    Friday

    Saturday

    1

    • mini-prep of cultivated G2’ E. coli

    2

    3

    4

    5

    6

    7

    8

    9

    • Digestion: G1 [ES] & G2‘ [XP] & pSB1C3 [EP]

    • ligation : insert G1[ES] & G2‘[XP]/vector pSB1C3[EP]

    10

    • strain test: activation of different strains overnight

    11

    • transfer to new medium, OD0.2 IPTGinduction, culture in 37゜C

    12

    • Single colony isolation from G1+G2’ LB-C plate, and cultivation in liquid LB-C

    • transfer to 27゜C

    13

    • mini-prep of cultivated G1+G2’ E. coli

    14

    15

    • Digestion: Ptet+B0032[ES] & GliI+KivD[XP]

    • sample and do GC

    16

    • ligation : insert Ptet+B0032[ES] & GliI+KivD[XP]/vector pSB1K3[EP]

    • Single colony isolation from G1+G2 LB-C plate, and cultivation in liquid LB-C

    17

    • transformation of Ptet+B0032+ GliI+KivD, and cultivation on LB- K plate

    • mini-prep of cultivated G1+G2 E. coli

    18

    • PCR of insert fragment [Ptet+B0032+ GliI+KivD]-----OK

    • DNA sequencing------NOT OK

    19

    20

    21

    22

    23

    24

    • carbon source test: activation DH5α overnight

    25

    • transfer to new medium(1/100), OD0.2 start counting culture time

    26

    • culturing for 72hours, inject feeding solution per 24hours

    27

    • culturing for 72hours, inject feeding solution per 24hours

    28

    • transformation of DNA program, and cultivation on LB- A plate

    • culturing for 72hours, inject feeding solution per 24hours

    29

    • ligation : insert Ptet+B0032[ES] & GliI+KivD[XP]/vector pSB1K3[EP]

    • Single colony isolation from DNA program LB-C plate, and cultivation in liquid LB-C

    • sample & do GC

    30

    • transformation of Ptet+B0032+ GliI+KivD, and cultivation on LB- K plate

    • mini-prep of cultivated DNA program E. coli

    • Do GC

    31

    • PCR of insert fragment [Ptet+B0032+ GliI+KivD] OK

    • DNA sequencing NOT OK

    Retrieved from "http://2012.igem.org/Team:NCTU_Formosa/Team"