Team:Fudan China/Timeline

HP

Month Events
May BUDS, a conference for initiators of iGEM teams
June E-mailing researchers in biological synthesis
Collecting public ideas of a sequential memory machine
July A lecture to the students attending biology summer camp
Delivering the idea of synthetic biology at a primary school in Anhui province, China
The 5th Asia-pacific iGEM conference, the idea of developing a theory to improve the presenting of iGEM project
August Developing the ‘iGEM taxonomy’
Developing the 3-D printed brick toy
FABO open night, The iGEM conference at the community
CCiC, Applying the theory to the presenting of our own Project
September Bringing our project and iGEM to the club fair
October The visit to the city sewage plant
Some lab work with the sample we tool from the sewage plant

Experiments

Overview

We began our project early in this year, but wasted lots of time on molecular cloning. We were making mistakes and debugging during most of the time. In July, we tried to build the memory sequential structures at first but failed at last because of leakage. Later, orthogonality of five integrases we have was tested. At the same time, we were tring to change promoter in our two-signal system and still working on this now. Furthermore, we have engaged in InterLab in the mid-summer. Efficiency of five integrases was tested in late September and October.

Details

Timeline Table (Summer only):

Date Order Module Experiment Result
Before July 1 Obtain 5 integrases used in our project Succeeded
2 Try to build a two-signal system(to examine the feasibility) Succeeded in building, but encountered with the problem of severe leakage of PLac
7/2 1 TSS Transform: 2 PLac parts, pSB1C3-R0010 & pSB1C3-R0011
7/3 1 TSS Plasmid Extraction: pSB1C3-R0010 & pSB1C3-R0011 Succeeded; confirmed by gel electrophoresis
2 TSS Digest: pSB1C3-R0010 & pSB1C3-R0011 using XbaI & PstI; gel electrophoresis and gel extraction Failed; too small digest products
7/4 1 TSS PCR: amplifying R0010 & R0011 Failed; no obvious bands in gel electrophoresis
7/5 1 TSS Replicate experiment 1 on 7/4 and change the annealing temperature; gel electrophoresis and gel extraction Succeeded
2 TSS PCR: prepare ssBxb1(previously-built TSS) backbone to ligate with R0010 & R0011; gel electrophoresis and gel extraction Succeeded
3 TSS In Fusion: ssBxb1 backbone(without promoter) + R0010 & R0011; transform; cultivate on Kana plate
7/6 1 TSS Single-colony cultivation: experiment 3 on 7/5; choose 5*2
7/7 1 TSS Plasmid Extraction: experiment 1 on 7/6; Digest with ApaI & BcuI; gel electrophoresis Succeeded, but still cannot confirm the ligation of R0010 or R0011
2 TSS PCR: confirming the ligation of R0010 or R0011 Failed, still cannot confirm the ligation of R0010 or R0011
7/10 1 TSS Sequencing: products of experiment 1 on 7/7 R0010: Succeeded; R0011: Failed.
7/11 1 IL Transform test devices to competent DH5alpha
7/12 1 IL Cultivate testing cell
7/13 1 TSS Testing: induce ssBxb1 using Arabinose Failed
2 TSS Plasmid Extraction: ssBxb1-R0010(to recover the attB/P sites)
3 TSS Digest: ssBxb1-R0010 & ssBT1(ssBxb1 without Bxb1 related parts) using ApaI & BcuI; gel electrophoresis and gel extraction Succeeded
4 TSS Ligation: digested ssBxb1-R0010 & ssBT1; transform; cultivate on KanaAmp plate
5 TSS Testing: replicate experiment 1 Failed
6 IL Drawing standard curves and cell meausurement Succeeded
7/14 1 TSS Colony PCR: plate of experiment 4 on 7/13 (test which clone does not leak) Correct clone: 1, 5, 6 (but it is confirmed false at last)
2 TSS Cultivate 1, 2, 5 in experiment 1
7/15 1 TSS Plasmid Extraction: experiment 2 on 7/14
2 TSS Sequencing: experiment 1 Clone 2 is correct; R0010 seems to leak as well
7/16 1 TSS Cultivate TF ssBxb1-R0010-1 & TF ssBxb1-R0010-5
7/17 1 TSS Plasmid Extraction: experiment 1 on 7/16
2 TSS Digest: experiment 1 using MluI; gel electrophoresis Failed; disturbed by TF
3 TSS Testing: induce TF ssBxb1-R0010-1 & TF ssBxb1-R0010-5 with Arabinose Failed
4 TSS Replicate the ligation and transform of experiment 3 on 7/13
7/18 1 TSS PCR: testing the sites of TF ssBxb1-R0010-1 Failed
7/19 1 TSS PCR: testing the sites of TF ssBxb1-R0010-1 Failed
2 TSS PCR: testing the phiBT1 attB/P sites Failed
3 TSS Testing: induce TF ssBxb1-R0010-1 & TF ssBxb1-R0010-5 with IPTG Failed
7/20 1 TSS Testing: in vitro experiment to validate the attB/P site of phiBT1 Succeeded
2 TSS PCR: obtain TF ssBxb1-R0010-1 backbone(to change the promoter) Failed
7/21 1 TSS Colony PCR: plate of TF ssBxb1-R0011 All except clone 8 are correct
7/22 1 TSS Try to optimize the condition of PCR Succeeded
2 TSS PCR: obtain TF ssBxb1-R0010-1 backbone(to change the promoter) & R0011 Succeeded
3 TSS In Fusion: TF ssBxb1-R0010-1 backbone(to change the promoter) + R0011; transform; cultivate on plate
4 O Colony PCR: plate of 5att-PBAD Failed
5 O Replicate experiment 4 with different primer pair Clone 2,4,5,6,7 are correct
6 O Cultivate clone 2,4,5,6,7 in experiment 5
7/23 1 TSS Colony PCR: plate of experiment 3 on 7/22 Clone 2,4 is correct
2 TSS Cultivate clone 2,4 in experiment 1
3 O Plasmid extraction: experiment 6 on 7/22; gel electrophoresis Larger than expected
4 O Sequencing: clone 2,4,5 False
7/24 1 TSS Plasmid extraction: ssR0011-2 & ssR0011-4; gel electrophoresis Correct
2 TSS PCR: prepare template for sequencing Succeeded
3 O PCR: Obtain 5 integrases and 5att-PBAD backbone Succeeded in obtain 5 integrases
4 O PCR: 5att-PBAD backbone Failed
7/25 1 TSS Sequencing: PCR product of ssR0011-2 & 4 False; original part is inconsistent
2 TSS In Fusion: ssEmpty + phiBT1 lin; transform
3 O Sequencing: att-PBAD False
7/26 1 TSS Colony PCR: experiment 2 on 7/25 Clone 2 is correct
2 TSS PCR: obtain R0011; gel extraction Succeeded
3 TSS In Fusion: 4H5 + ssBxb1 backbone; transform
7/27 1 TSS Make competent cell TF(TOP10 with transcriptional factors)
2 TSS Sequencing: ssBT1-2-2 Correct
3 TSS Testing: Bxb1 sites of ssR0011-4 Totally inverted
4 TSS Cultivate ss4H5 1,4,5,6,8
7/28 1 TSS Sequencing: 4H5 1,4,5 False
2 TSS Plasmid extraction: 4H5 1
3 TSS Digest: 4H5 1 using ApaI & BcuI; gel extraction Succeeded
4 TSS T4 ligation; transform
7/29 1 TSS Colony PCR: experiment 4 on 7/28 2, 3, 4 are correct
2 TSS Testing: Bxb1 sites of 2, 3, 4 Inverted
8/2 1 TSS PCR: ssBxb1 lin Failed
2 TSS PCR: ssEmpty lin Failed
3 TSS Anneal to obtain R0011
4 O PCR: Obtain 5 integrases with B0033; gel extraction Succeeded
5 O PCR: Obtain 5att backbone Failed
8/3 1 TSS PCR: ssBxb1 lin & ssEmpty lin Failed
2 TSS PCR: replicate 1 & I759016 Succeeded in ssEmpty & TetR
3 TSS PCR: ssBxb1 lin
4 TSS Cultivate TF
5 TSS Digest: TetR using EcoRI & BcuI; pUC19 AraC-2 using XbaI & EcoRI
6 TSS T4 ligation: TetR + pUC; transform
7 TSS In Fusion: ssEmpty lin + phiBT1 lin; transform Inverted
8 O PCR: 5att-PBAD Succeeded
9 O In Fusion: 5 integrases + backbone; transform
8/4 1 TSS Colony PCR: experiment 7 on 8/3 Failed
2 TSS Sequencing: BT1-1 & BT1-2
3 TSS PCR: ssBxb lin Failed
4 TSS Replicate experiment 3 Succeeded
5 TSS Replicate experiment 6 on 8/3
6 TSS In Fusion: ssR0011 *2; transform
7 TSS Cultivate BT1 1/2
8/5 1 TSS Replicate experiment 6 on 8/4; condensed
2 TSS Plasmid extraction: experiment 7 on 8/4; SapI & SspI digest False
3 TSS Colony PCR: experiment 7 on 8/3 1 is correct
4 TSS Colony PCR: experiment 5 on 8/4 All are false
5 TSS PCR: 8/4 4, gel extraction Succeeded
6 O Colony PCR: 5PR-int Succeeded
8/6 1 TSS Colony PCR: experiment 5 on 8/4 1, 15 are correct
2 TSS Plasmid extraction: ssR0011-1-1; Digest using ApaI & SapI False
3 TSS Sequencing: ssBT1-2-3 on 8/3 False
4 O Testing: orthogonal of 5 integrases make sense
8/7 1 TSS Plasmid extraction: experiment 2, 6 on 8/6; digest: ss0011 1-2 using SapI & SspI
2 O Replicate experiment 4 on 8/6 make sense
8/8 1 TSS In Fusion: R0011 lin(8.2) + 8.5 1,2; transform
2 TSS Digest: TetR using EcoRI & BcuI; TF using EcoRI & XbaI; gel extraction Succeeded
3 TSS T4 ligation: TetR di + TF di; transform
4 O Replicate experiment 4 on 8/6; and optimize the conditions Succeeded
8/9 1 TSS Colony Culture: experiment 1 on 8/8; Plasmid extraction; Digest using ApaI & ApaLI False
2 TSS Colony Culture: experiment 3 on 8/8
8/10 1 TSS Plasmid extraction: experiment 2 on 8/9; digest using ApaI & ApaLI 1, 5 are correct
8/11 1 TSS PCR: ss0011 1-1 & ss0011 1-2; gel extraction Succeeded; low concentration
2 TSS Digest: experiment 1 using EcoRI + PstI False
2 TSS Digest: TF using BcuI & PstI; TetR using XbaI & PstI; gel extraction Succeeded
3 TSS T4 ligation: TF di + TetR di; transform
4 TSS In Fusion: ss0011 + ss0011 1-2; transform
8/12 1 TSS Colony Culture: experiment 3, 4 on 8/11; Plasmid extraction; Digest: pUCTet using MluI & PstI and ss0011 using ApaI & ApaLI Tet: 1-5; 0011: 1-2, 1-4; 2-2, 2-4 are correct
8/14 1 M PCR: mutate Bxb1-1 & phiBT1-1&2; gel extraction Succeeded
2 M PCR: mutate phiC31; gel extraction Succeeded
3 M In Fusion: phiBT1 m1 + phiBT1 m2 & phiC31 m1 + phiC31 m2; transform
4 M Digest: Bxb1 m using DpnI; transform
8/15 1 M Colony Culture: experiment 3, 4 on 8/14; Digest using EcoRI & XbaI All are correct
8/16 1 M PCR: mutate Bxb1-2&3; gel extraction Succeeded
2 M In Fusion: Bxb1 m2m3 1 + 2; transformation
8/17 1 M Colony Culture: expriment 2 on 8/16; Digest using XbaI & PstI All are correct
2 TSS Culture: ss0011 2-4 & ssBT1-3; Digest XbaI & SspI; gel extraction; T4 ligation; transform
8/18 1 TSS Culture: ss0011 2-4 & ssBT1-3; Digest ApaI & HindIII; gel extraction Succeeded; low concentration
2 TSS Replicate experiment 1
8/19 1 TSS Culture: ssBT1-3; Plasmid extraction; Digest ApaI & HindIII & NruI; gel extraction Succeeded
2 TSS T4 ligation: ss0011 + ssBT1; transformation
8/20 1 TSS Colony Culture: experiment 2 on 8/19
2 M PCR: mutate Bxb1 m3; gel extraction Succeeded
3 M In Fusion: Bxb1 m4 1 + 2； transform
8/21 1 M Colony Culture: experiment 3 on 8/20; Sequencing
2 E Obtain 1PR-BT1 & 1PR-C31
3 TSS Plasmid extraction: experiment 1 on 8/20; Digest using KpnI & SspI Correct
4 TSS Testing: induce ss0011 with IPTG Failed
8/22 1 TSS PCR: testing ssBT1-3 Failed
8/23 1 E Testing: 1PR-BT1 & 1PR-C31 Correct
2 TSS Plasmid extraction: ssBT1&3; Digest ssBT1-3 using ApaLI & HindIII, ApaI & ApaLI, ssBT1-1 using ApaLI & HindIII; gel extration Succeeded
3 TSS T4 ligation: experiment 2; transform
4 E Testing: induce 1PR-BT1 & 1PR-C31 with arabinose
8/24 1 E PCR: Rv1 & TG1 & Bxb1 lin; gel extraction Succeeded
2 E In Fusion: Rv1 & TG1 del frg + RvTG pls; transform
3 TSS Colony PCR: ssBT1-4 & ss0011 TF Only 5TF 4 is correct
8/25 1 TSS Plasmid extraction: ssBT1-4; Digest using KpnI & SspI 2, 4 are correct
2 E Colony PCR: 1PR-Bxb1 TF/5TF TF1 and 5TF 5, 6 are correct
3 E Colony Culture: experiment 2 on 8/24
4 TSS Testing: ss0011 5TF 4
8/26 1 TSS Colony PCR: ss0011 TF 7 is correct
2 TSS Testing: induce ss0011 5TF 4 with arabinose Failed
3 E Plasmid extraction: experiment 3 on 8/25; Digest using E & P All except Rv1 3 are correct
4 E T4 ligation: TG1/Rv1 del EP di + TG1/Rv1 EB di + PBAD; transform
8/27 1 E Colony PCR: experiment 4 on 8/26 Rv1 15, 16 ,TG1 14 are correct
8/28 1 TSS Plamid extraction: ss0011 4-7; Digest using ApaI & HindIII Failed
2 TSS T4 ligation: replicate experiment 3 on 8/23
After September 1 E Test the efficiency of 5 integrases by qPCR
2 Wrap all the parts for shipping

Abbreviations: TSS, Two-signal system; IL, InterLab; O, Orthogonality of integrases; M, Mutation of integrases; E, Efficiency of integrases.