In the notebook below, we give the following abbreviation:
1.Template Plasmid
G001: pSSTG1
G002: pSSTG2
pRGm1/m2/m3: pRG with different promotors
2. Report Plasmid
Naming format: G001/G002-IC35/IF55/IS37/E21/T25/G22
G001/G002: the template plasmid G001, G002
IC35/IF55/IS37/E21/T25/G22: the corresponding recombinase recognition sites and the terminators within
IC35: phiC31-ECK120034435
G22: TG1-L3S3P22
IF55: INT5-ECK120010855
IS37: INT7-ECK120033737
IE21: INT8-ECK120030221
T25: INT10-ECK120020525
3. Testing Strain
Naming format: G1/G2+ IC35/IF55/IS37/E21/T25/G22+A1/A2/A4
G1/G2: the template plasmid G001, G002
IC35/IF55/IS37/E21/T25/G22: same as the naming in report plasmid
A1/A2/A4: RBSs with different translation strength
4. Chromoprotein expression sequence
R: Red by expression of eforRed
Y: Yellow by expression of amilGFP
B: Blue by expression of amilCP
5. Flipping System
pRG-Recombinase 1-Recombinase 2v
A flipping system with 3 different states by using 2 different recombinases
Weekly Progress
Week One
14/07/2019-20/07/2019
1. Set up the project and confirm the project topic.
Week Two
21/07/2019-27/07/2019
1. Design six [attB-Terminator-attP] sequences:
phiC31-ECK120034435
TG1-L3S3P22
INT5-ECK120010855
INT7-ECK120033737
INT8-ECK120030221
INT10-ECK120020525
Send the sequences to synthesis.
2. Design plasmids pSSTG1(G001), pSSTG2(G002) as testing platform. Design the construction plan and primers.
Naming format: G001/G002-IC35/IF55/IS37/E21/T25/G22
G001/G002: the template plasmid G001, G002
IC35/IF55/IS37/E21/T25/G22: the corresponding recombinase recognition sites and the terminators within
IC35: phiC31-ECK120034435
G22: TG1-L3S3P22
IF55: INT5-ECK120010855
IS37: INT7-ECK120033737
IE21: INT8-ECK120030221
T25: INT10-ECK120020525
3. Testing Strain
Naming format: G1/G2+ IC35/IF55/IS37/E21/T25/G22+A1/A2/A4
G1/G2: the template plasmid G001, G002
IC35/IF55/IS37/E21/T25/G22: same as the naming in report plasmid
A1/A2/A4: RBSs with different translation strength
4. Chromoprotein expression sequence
R: Red by expression of eforRed
Y: Yellow by expression of amilGFP
B: Blue by expression of amilCP
5. Flipping System
pRG-Recombinase 1-Recombinase 2v
A flipping system with 3 different states by using 2 different recombinases
Weekly Progress
Week One
14/07/2019-20/07/2019
1. Set up the project and confirm the project topic.
Week Two
21/07/2019-27/07/2019
1. Design six [attB-Terminator-attP] sequences:
phiC31-ECK120034435
TG1-L3S3P22
INT5-ECK120010855
INT7-ECK120033737
INT8-ECK120030221
INT10-ECK120020525
Send the sequences to synthesis.
2. Design plasmids pSSTG1(G001), pSSTG2(G002) as testing platform. Design the construction plan and primers.
R: Red by expression of eforRed
Y: Yellow by expression of amilGFP
B: Blue by expression of amilCP
5. Flipping System
pRG-Recombinase 1-Recombinase 2v
A flipping system with 3 different states by using 2 different recombinases
Weekly Progress
Week One
14/07/2019-20/07/2019
1. Set up the project and confirm the project topic.
Week Two
21/07/2019-27/07/2019
1. Design six [attB-Terminator-attP] sequences:
phiC31-ECK120034435
TG1-L3S3P22
INT5-ECK120010855
INT7-ECK120033737
INT8-ECK120030221
INT10-ECK120020525
Send the sequences to synthesis.
2. Design plasmids pSSTG1(G001), pSSTG2(G002) as testing platform. Design the construction plan and primers.
Figure 1: the structure of plasmid G001
Figure 2: the structure of plasmid G002
3. Construct the induced-expressive plasmid (G003) of recombinase phiC31 on the basis of plasmid pRG.
Week Three
28/07/2019-03/08/20191. Do the human practice in Shenzhen Children’s Hospital
2. Construct the induced-expressive plasmids (G004-G008) of recombinases INT5, INT7, INT8, INT10, TG1 on the basis of plasmid pRG.
3. Mutate the sequence of -10 region in the pTAC promotor of the plasmid pRG, and construct the induced-expression devices pRGm1, pRGm2, pRGm3 which have different dynamic ranges.
4. Construct plasmids G009 and G010 on the basis of G001 and G002, respectively.
Week Four
04/08/2019-10/08/20191. Construct the report plasmid (G001-IC35/E21/T25/G22) on the basis of plasmid G001.
2. Construct the report plasmid (G001-IC35/E21/T25/G22) on the basis of plasmid G002.
3. Culture the testing strains G1IC35A1, G2IC35A1, G1IT25A1, G2IT25A1, G1IG22A1, G2IG22A1, G1IE21A1, G2IE21A1.
4. Assemble sfGFP to pRGm1, pRGm2, pRGm3.
Week Five
11/08/2019-17/08/20191. Apply qualitative test to strains G2IC35A1, G2IT25A1, G2IG22A1, G2IE21A1 and determine the validity of the recombinase expressing system as well as the state effect the reversal of terminator has on the downstream gene.
2. By flow cytometry, quantitatively test the relay devices constructed by recombinase phiC31 and INT10 (G2IC35A1, G2IT25A1). Prove the feasibility of the inducing system and confirm the dynamic ranges of the terminator switches.
Week Six
18/08/2019-24/08/2019Prepare and take part in CCiC (Conference of China iGEMer Community).
Week Seven
25/08/2019-31/08/20191. Use steady-state method to test the biological relays G1IC35A1, G1IT25A1, G1IG22A1, G1IE21A1.
2. Standardize the intensity of the systems’ input signal by IPTG-gradient inducing pTAC-sfGFP through the steady-state method.
3. Apply simplified test on the biological relays G2IC35A1, G2IT25A1, G2IG22A1, G2IE21A1.
4. Construct the report plasmid (G001-IF55/IS37) on the basis of plasmid G002.
Week Eight
01/09/2019-07/09/20191. Culture the testing strains G2IF55A1, G2IS37A1.
2. Qualitatively test G2IF55A1, G2IS37A1.
3. Assemble eforRed to G002-IE21, construct the plasmid G2IE21-R1
Assemble amilGFP to G002-IT25, construct the plasmid G2IE21-Y1
Assemble amilCP to G002-IC35, construct the plasmid G2IE21-B1
Week Nine
08/09/2019-14/09/20191. Find the insufficiency of the chromoprotein expression level in plasmids G2IE21-R1，G2IC35-B1. Optimize the RBS of the plasmids.
2. Construct the induced-expression system which combines recombinase phiC31, INT8, INT10, TG1 with RBS component B0032, B0034, on the basis of pRG.
Week Ten
15/09/2019-21/09/20191. Culture the testing strains G2IC35A2/A4, G2IT25A2/A4, G2IE21A2/A4, G2IG22A2/A4, and apply qualitative test on them.
2. Do human practice in Huaqiang North electronic market
3. Reconstruct the testing strains G2IC35A1, G2IT25A1, G2IE21A1, G2IG22A1
Week Eleven
22/09/2019-28/09/20191. Repeat the simplified test for G2IC35A1, G2IT25A1, G2IG22A1, G2IE21A1
2. Use steady-state method to test the gradient-induced curve for pRGm1-sfGFP、pRGm2-sfGFP、pRGm3-sfGFP
3. Do public engagement in Shenzhen Middle School
4. Categorize the part
Week Twelve
29/09/2019-05/10/20191. Build up the color report system G010-BYR, G010-RYB on the basis of G010
2. Build up the flipping system pRG-IC1-T1, pRG-T1-IC2, pRG-T1-E1 on the basis of pRG
3. Change a weak promotor for INT8
4. Start to make Wiki
Week Thirteen
06/10/2019-12/10/20191. Repeat the quantitative inducing experiment for G2IC35A1, G2IT25A1, G2IG22A1
2. Test and debug two ADC (analog-digit converter) system
3. Perfect the computer program
Week Fourteen
13/10/2019-19/10/20191. Do public engagement in Shenzhen Media Group
2. Complete the judging form
3. Finish Wiki
