Difference between revisions of "Team:Tongji China/Record"

Plasmid Construction Parts (Week 1-6: June 5-July 10)

Week 1

June 5
pUAST-3xHA transform into E.coli.
June 6
1. Pick up the single colony, and culture it with 10 mL 2xYT medium overnight.
2. Design sequencing primers of pUAST-3xHA.
June 7
1. Midi extraction of plasmids pUAST-3xHA.
2. Send it to GENEWIZ to sequence.
June 8
1. Verify the sequencing result, it is correct.
2. Digest pUAST-3xHA with BamHI overnight.
June 9
Extraction of Drosophila genome and RNA, purify the digested vector.

Week 2

June 12
Reverse transcription of Drosophila RNA, then get the cDNA.
June 13
Design the primers for fragments’ PCR and fragments’ sequencing.
June 14
1. Clone pleP, GAL4, GAL80ts from genome (success) and TH from cDNA (failed), purify them.
2. Send them to GENEWIZ to sequence.
June 15
1. Verify the sequencing result, it is correct.
2. Ligase each of them with pLB vector and transform into E.coli.
3. Clone TH from cDNA again (failed).
June 16
1. Pick up the single colony, do bacteria liquid PCR.
2. Send 5 positive colony to GENEWIZ to sequence.
3. Design the In-Fusion primers.
4. Clone TH from cDNA again (failed).

Week 3

June 19
1. Verify the sequencing result, it is correct.
2. pleP, GAL4, GAL80ts PCR with In-Fusion primers, then purify them.
3. Design new primers of TH PCR.
June 20
1. In-Fusion cloning: pUAST-pleP-GAL4, pUAST-pleP-GAL80ts, and then transform into E.coli.
2. Clone TH from cDNA again (failed).
June 21
1. No colony. Do In-Fusion and transform again.
2. Clone TH from cDNA again (failed).
3. Design new MCS for pUAST BamHI.
June 22
1. Pick up the single colony, do bacteria liquid PCR, they are all negative.
2. Extraction of Drosophila RNA again, reverse transcription of it, then get the cDNA.
3. Ligase MCS with pUAST BamHI, then transform.
June 23
1. Pick up the single colony (pUAST-new MCS), do bacteria liquid PCR, and send 5 of positive colony to GENEWIZ to sequence.
2. Clone TH from cDNA again (failed).
3. Design a pair of new primer for pleP’ single digestion.
4. Do In-Fusion and transform again.

Week 4

June 26
1. No colony for In-Fution.
2. Verify the sequencing result (pUAST-new MCS), it is correct.
3. Culture one of the correct colony overnight.
4. Clone TH from cDNA again (failed).
5. pleP PCR with single digestion primer, then purify it.
6. Design three pair of new primer for In-Fusion Cloning.
June 27
1. Place a synthesis order for TH.
2. Midi extraction of plasmids pUAST-new MCS.
3. pleP, GAL4, GAL80ts PCR with In-Fusion primers, then purify them and send them to GENEWIZ to sequenced.
4. Digest pUAST-new MCS and pelP PCR purified product by BglII.
5. Purify digested pleP PCR product and pUAST-new MCS.
6. Ligase them and transform into E.coli.
June 28
1. Pick up the single colony (pUAST-pleP), do bacteria liquid PCR, and send the positive colony to GENEWIZ to sequence.
2. Verify the sequencing result (In-Fusion fragment: GAL4, GAL80ts), it is correct.
3. Design two pair of primers for GAL4 and GAL80ts’ double digestion.
June 29
1. Verify the sequencing result (pUAST- pleP), it is correct.
2. Do In-Fusion cloning again.
3. GAL4 and GAL80ts PCR with double digestion primer, then purify it.
4. Culture one of the correct colony overnight.
June 30
1. Midi extraction of plasmids pUAST-pleP.
2. Digest pUAST-pleP, GAL4 and GAL80ts PCR purified product by EcoRI and HpaI.
3. Purify digested pUAST-pleP, GAL4 and GAL80ts PCR product.
4. Ligase pUAST-pleP with GAL4, pUAST-pleP with GAL80ts, and transform into E.coli.
5. No colony for In-Fution.

Week 5

July 3
1. Pick up the single colony (pUAST-pleP), do bacteria liquid PCR, they are all negative.
2. Ligase pUAST-pleP with GAL4, pUAST-pleP with GAL80ts, and transform into E.coli again.
July 4
1. No colony for In-Fusion.
2. Pick up the single colony (pUAST-pleP-GAL4, pUAST-pleP-GAL80ts), do bacteria liquid PCR, they are all negative.
July 5
Do In-Fusion and transform again.
July 6
1. Pick up the single colony (In-Fusion: pUAST-pleP-GAL4, pUAST-pleP-GAL80ts), do bacteria liquid PCR, and send five of the positive colony to GENEWIZ to sequence.
2. Test and verify pUAST-TH, pUAST-pleP-GAL4 and pUAST-pleP-GAL80ts by single digestion. Positive.
July 7
1. Verify the sequencing result (In-Fusion: pUAST-pleP-GAL4, pUAST-pleP-GAL80ts), it is correct.
2. Each of one culture one of the correct colonies overnight.
3. Receive the synthesis pUAST-TH from .

Week 6

July 10
Maxi extraction of plasmids pUAST-pleP-GAL4, pUAST-pleP-GAL80ts.

Micro-injection (July-August)

We ask the SIBS (Shanghai Institute for Biological Sciences) to inject the plasmid into Drosophila egg using micro-injection technology.

Screening (September)

After microinjection, we screen the transgenic flies.

Behavior experiment (Week 1-4: October 4- October 25)

Week 1

October 4
1. Start experiment 7: Draw the reproductive curve of the modified fruit fly.
2. Part of experiment 6: Are the viability of modified fruit flies affected (Lack food and water)?
October 5
Record the number of eggs laid by females in experiment 7(the first day).
October 6
1. Record the number of eggs lay by females in experiment 7(the second day).
2. Experiment 4: Test whether the modified fruit flies increase its appeal to males.
October 7
1. Record the number of eggs lay by females in experiment 7(the third day). (One fly died because of artificial operation so we start the experiment again)
2. Experiment 3: Test the modified Drosophila’s gender preferences.

Week 2

October 8
1. Experiment 2: Detect the male-male courtship when raising the temperature.
2. Repeat experiment 3.
October 9
1. Start experiment 7: ‘Draw the reproductive curve of the modified fruit fly’ again.
2. As the eggs do not necessarily hatch, we decide to wait a few days to count the larvae.
October 10
Transfer the female flies to the new culture tubes (experiment 7).
October 11
1. Transfer the female flies to the new culture tubes (experiment 7).
2. Experiment 5: Does the co-culture of wild fruit flies and modified fruit flies influence their courtship of wild-type females?
October 12
Transfer the female flies to the new culture tubes (experiment 7).
October 13
Transfer the female flies to the new culture tubes (experiment 7).
October 14
1. Transfer the female flies to the new culture tubes (experiment 7).
2. Part of experiment 6: Are the viability of modified fruit flies affected (high temperature)?
3. Experiment 1: Use Real-time PCR to detect whether the expression of TH is increased at 29℃.

Week 3

October 15
1. Record the number of eggs laid by females in experiment 7(the first day).
2. Transfer the female flies to the new culture tubes (experiment 7).
3. Part of experiment 6: Are the viability of modified fruit flies affected (the seismo-tube experiment)?
October 16-21
1. Complete experiment 7.
2. Complete experiment 8.

Week 4

October 22-25
Processing data obtained from the experiment.

March

March 10
Learn about the bio-safety measures and experimental method in professor Du, Gao and professor Xue’s Lab.
March 20
Passed the experiment and bio-safety test and obtains qualifications.

April

April 8
Hold the first OPEN NIGHT LIVE.
April 15
Visit to Shanghai CDC and Yangpu Hospital Affiliated to Tongji University.

May

May 14
Make friend with SDU-iGEM and gave the bio-brick BBa_K1922003(wt p53) and BBa_K1922003(htERT-p53) to them.
May 19
Hold Campus talk about iGEM and synthetic biology in Tieling High School.

June

June 9
Do a questionnaire to understand what the children and parents want in the STEAM course.
June 15
Analyze the data of questionnaire and start to design synthetic biology STEAM course.

June

June 9
Do a questionnaire to understand what the children and parents want in the STEAM course.
June 15
Analyze the data of questionnaire and start to design synthetic biology STEAM course.

June

June 9
Do a questionnaire to understand what the children and parents want in the STEAM course.
June 15
Analyze the data of questionnaire and start to design synthetic biology STEAM course.

July

July 21
Finish the STEAM course design and start to design bio-toolbox with the help of Fablab Shanghai.
July 22
Start to design the board game ‘BIO-CREATOR’.

August

August 16
Take part in CiCC held by FAFU and exchange our project with other iGEM teams in China.
August 19
Hold OPEN NIGHT 2ND LIVE and invite the team Fudan, Fudan_China, Shanghaitech, STJU_BioX_Shanghai and ECUST.

September

September 12
Give class to students from other major in Tongji University in elective course.
September 20
Finish the bio-toolbox design and start the industrial design drawing.

October

October 7
Take part in the synthetic biology creative competition hold by LANJING Biology Company.
October 14
Make the bio-toolbox.
October 16
Launch a propaganda campaign of iGEM and synthetic biology.
Start recruiting activities.