Team:ZJU-China/Notebook

Notebook
June

6.15
        Participant: Zhanyu Wang, Han Wan
        1.Get all the basic parts from Kit plate
        supD-tRNA ,T7RNAP &PT7
Transformation
Gene of interest Vector Ice 42°C Ice 37°C+LB Plate
BBa_K228001 pSB1C3 30min 90s 5min 1h/1ml LBCM
BBa_K228000 pSB1C3 30min 90s 5min 1h/1ml LBCM
BBa_K1321338 pSB1C3 30min 90s 5min 1h/1ml LBCM
        2.Done the transformation
6.30
        Transformation
        Participant： Shisheng Li
Transformation
Gene of interest Vector Ice 42°C Ice 37°C+LB Plate
BBa_I15008 pSB1C3 30min 90s 5min 1h/1ml LBCM
BBa_I15009 pSB1C3 30min 90s 5min 1h/1ml LBCM


July

    
7.1
        Liquid Culture
        Participant: Shisheng Li
Liquid Culture
Name Medium Volume/ml
BBa_I15008(pSB1C3) colony_1 LB 10
BBa_I15008(pSB1C3) colony_2 LB 10
BBa_I15008(pSB1C3) colony_3 LB 10
BBa_I15009(pSB1C3) colony_1 LB 10
BBa_I15009(pSB1C3) colony_2 LB 10
BBa_I15009(pSB1C3) colony_3 LB 10

7.2

        miniprep
        Participant: Shisheng Li
Miniprep
Name 260/280 C ng/μl
BBa_I15008(pSB1C3) colony_1 1.90 34.6
BBa_I15008(pSB1C3) colony_2 1.87 33.2
BBa_I15008(pSB1C3) colony_3 1.88 58.3
BBa_I15009(pSB1C3) colony_1 1.75 33.0
BBa_I15009(pSB1C3) colony_2 1.85 45.1
BBa_I15009(pSB1C3) colony_3 1.93 49.0
        PCR
        Participant： Shisheng Li
PCR V/μL Steps Temperature/μL Time/s Cycle
taq polymerase 0.5 Pre Denature 94 180 /
tempalte 0.5 Denature 95 30 30
10X Taq buffer 5 Annealing 55 30 30
primer-forward(10μM) 2 Extension 72 45/45 30
primer-reverse(10μM) 2 Final Extension 72 300 /
dNTP(2.5mM) 4 storage 4 / /
ddH2O 36 / / / /
    Result:

7.5
        Construct of double oscillation plasmid Ptd103aiiA-luxS
        Participant：Jinshi Ran, Hongya Zhu
        • We got luxS gene from 15M, plate 6, distribution kit 2016.
        • We amplified luxS with PCR and added sequences homo with pTD103aiiA to it.
        • We amplified the backbone pTD103aiiA, and added sequence homo with luxS.
        • Using one-step cloning, we assembled them together.
        • We transformed the new constructed plasmid into DH5alpha and cultured it overnight.
        • We screened the positive colony with colony PCR. After conformed, we inoculated it in 5ml LB and cultured it overnight to harvest the pTD103aiiA-luxS.


7.8
        Transformation
        Participant：Shisheng Li
Transformation
Gene of interest Vector Ice 42°C Ice 37°C+LB Plate
cph8 pSB1C3 30min 90s 5min 1h/1ml LBCM
double terminator pSB1C3 30min 90s 5min 1h/1ml LBCM
PompC pSB1C3 30min 90s 5min 1h/1ml LBCM

7.9
        Liquid Culture
        Participant: Shisheng Li
Liquid Culture
Name Medium Volume/ml
Cph8 colony_1 LB 10
Cph8 colony_2 LB 10
double terminator colony_1 LB 10
double terminator colony_2 LB 10
PompC colony_1 LB 10
PompC colony_2 LB 10

7.10
        Miniprep
        Participant: Shisheng Li
Miniprep
Name 260/280 C ng/μl
Cph8 colony_1 1.90 53.3
Cph8 colony_2 1.87 74.1
double terminator colony_1 1.84 44.2
double terminator colony_2 1.61 50.8
PompC colony_1 1.87 43.8
PompC colony_2 2.12 37.6
        PCR
        Participant: Shisheng Li
PCR V/μL Steps Temperature/μL Time/s Cycle
taq polymerase 0.5 Pre Denature 94 180 /
tempalte 0.5 Denature 95 30 30
10X Taq buffer 5 Annealing 55 30 30
primer-forward(10μM) 2 Extension 72 180/45/45 30
primer-reverse(10μM) 2 Final Extension 72 300 /
dNTP(2.5mM) 4 storage 4 / /
ddH2O 36 / / / /
        Result:
        The result of gel electrophoresis showed that we managed to get the pcr product of Cph8 and PompC but failed in getting double terminator. After we changed the annealing temperature, we finally made it. When sorting out data, we accidently deleted the picture of the gel.

7.16

        1.Measuring and modeling of promoter pluxR
        Participant: Jinshi Ran, Hongya Zhu
        For better description of single oscillation system, we measured the response intensity of pluxR to AHL. First, we mutated pTD103luxI-sfGFP with knocking gene luxI out with PCR. We named the new plasmid pTD103sfGFP.
We transformed MG1655 strain of E•coil with pTD103sfGFP, inoculated positive colony in 5ml LB with antibiotic 100μg/ml kanamycin and cultured it over night.
We inoculated over night culture with a 1:1000 dilution in 1ml LB and added AHL according a concentration range:10-6~10-11M when the bacteria solution reached an A600nm 0.1.
Cultured it in 37℃ for 8h. We spun it down and concentrated in PBS (pH=7.2), then measured the fluorescence intensity.

        2.pick colonies and shake
        Participant：Zhanyu Wang, Han Wan
Liquid Culture
Name Medium Volume/ml
BBa_K28001(pSB1C3) colony_1 LB 10
BBa_K28001(pSB1C3) colony_2 LB 10
BBa_K28001(pSB1C3) colony_3 LB 10
BBa_K28000(pSB1C3) colony_1 LB 10
BBa_K28000(pSB1C3) colony_2 LB 10
BBa_K1321338(pSB1C3) colony_1 LB 10
BBa_K1321338(pSB1C3) colony_2 LB 10
BBa_K1321338(pSB1C3) colony_3 LB 10
7.26

        Find the T7ptag mutation site and design primers for point mutation
        Participant：Zhanyu Wang, Han Wan

7.27

        Point mutation pcr
        Participant：Zhanyu Wang, Han Wan


August

8.2
        Confirm other parts need in logic gate
        Participant：Wang Zhanyu, Wan Han
Transformation
Gene of interest Vector Ice 42°C Ice 37°C+LB Plate
BBa_R0010 pSB1C3 30min 90s 5min 1h/1ml LBCM
BBa_I13453 pSB1C3 30min 90s 5min 1h/1ml LBCM
BBa_895006 pSB1C3 30min 90s 5min 1h/1ml LBCM

8.3
        pick colonies and shake
        Participant：Participant：Zhanyu Wang, Han Wan
Liquid Culture
Name Medium Volume/ml
BBa_R0010(pSB1C3) colony_1 LB 10
BBa_R0010(pSB1C3) colony_2 LB 10
BBa_R0010(pSB1C3) colony_3 LB 10
BBa_I13453(pSB1C3) colony_1 LB 10
BBa_I13453(pSB1C3) colony_2 LB 10
BBa_K895006(pSB1C3) colony_1 LB 10
BBa_K895006(pSB1C3) colony_2 LB 10
BBa_K895006(pSB1C3) colony_3 LB 10
8.4

        Participant：Zhanyu Wang, Han Wan
        1. Extract the plasmid
        2. PCR
PCR V/μL Steps Temperature/μL Time/s Cycle
taq polymerase 0.5 Pre Denature 94 180 /
tempalte 0.5 Denature 95 30 30
10X Taq buffer 5 Annealing 55 30 30
primer-forward(10μM) 2 Extension 72 45/45 30
primer-reverse(10μM) 2 Final Extension 72 300 /
dNTP(2.5mM) 4 storage 4 / /
ddH2O 36 / / / /
        3.Conduct electrophoresis in 1% agarose gel
        After sequencing, the other parts are all confirmed.

8.5

        Doing overlap PCR, construct the logic gate plasmid.
        Participant：Zhanyu Wang, Han Wan

8.7-8.9

        Finish the construction of logic gate’s input plasmid(~3500bp,marked with an arrow)
        Participant： Zhanyu Wang, Han Wan/br>

8.9-8.11

        Finish the construction of logic gate’s output plasmid(~1100bp,marked with arrows)
        Participant： Zhanyu Wang, Han Wan


8.11-8.14

        Using infusion cloning to integrate the input plasmid and output plasmid together. (~4500bp,marked with an arrow)
        Participant： Zhanyu Wang, Han Wan


        Thus , we finally get our logic gate plasmid with input and output together.

8.15

        Validate the logic gate plasmid. For details, see our protocol in the experiment part.
        Participant： Zhanyu Wang, Han Wan

8.18

        Validate the logic gate's response concentration.
        Participant： Zhanyu Wang, Han Wan

        we add 0.5ml of the fresh bacteria culture and appropriate volume of inducer solution to prepare induction system with the concentration gradient of 10^-9, 10^-8, 10^-7, 10^-6, 10^-5, 10^-4, 10^-3.
        For details, see our protocol in the experiment part.
8.20

        Co-transformation
        Participant: Shisheng Li

Co-Transformation
Gene of interest Vector Ice 42°C Ice 37°C+LB Plate
Two green-TCS plasmids / 30min 90s 5min 1h/1ml LBCM
Two red-TCS plasmids / 30min 90s 5min 1h/1ml LBCM

8.21
        Liquid Culture
        Participant: Zhanyu Wang, Han Wan
Liquid Culture
Name Medium Volume/ml
Green TCS_1 LB 5
Green TCS_2 LB 5
Green TCS_3 LB 5
Green TCS_4 LB 5
Green TCS_5 LB 5
Green TCS_6 LB 5
Green TCS_7 LB 5
Green TCS_8 LB 5
Red TCS_1 LB 5
Red TCS_2 LB 5
Red TCS_3 LB 5
Red TCS_4 LB 5
Red TCS_5 LB 5
Red TCS_6 LB 5
Red TCS_7 LB 5
Red TCS_8 LB 5

8.22
        Fluorescence intensity measuring
        Participant: Shisheng Li
Green TCS
RFU Control Dark/th> green light red light
1 9074 31813 39406 32336
2 9022 31512 39105 32491
3 9063 31980 38535 32000
4 9072 32396 39816 32261
5 9130 32203 39201 33136
6 9063 32463 39829 33519
7 9329 32370 39991 33775
8 9192 32724 39886 33168

Red light TCS
RFU Control Dark/th> red light
1 369 3716 3241
2 248 3719 3332
3 384 3743 3263
4 404 3761 3449
5 408 3663 3179
6 409 3761 3416
7 350 3761 3416
8 357 3584 3178

8.23
        Verifying the single oscillation circuit in microfluidics device
        Participant：Jinshi Ran, Hongya Zhu
        • We co-transformed the MG1655 strain with pTD103aiiA and pTD103luxI-sfGFP
        • We screened the positive colony and inoculated in 50ml LB with antibiotic 100μg/ml ampicillin (Amp) and 50 mgml21 kanamycin (Kan).         • We spun it down and concentrated it in 5ml of fresh media with surfactant concentration of0.075% Tween20 when the bacteria solution reach an A600nm of 0.05~0.1.
        • We loaded the sample from the cell port while keeping the media port at sufficiently higher pressure than the waste port below to prevent contamination. We manually applied pressure pulses to line to induce a momentary flow change. The flow was then reversed and allow for cell to receive fresh media with 0.075% Tween20 which prevented cells from adhering to the main channels and waste ports.
        • We took pictures of microfluidics in fluorescence microscope every 5 mins and analyzed the fluorescent density with imageJ.


8.25
        Liquid Culture
        Participant: Shisheng Li
Liquid Culture
Name Medium Volume/ml
Green TCS_1 LB 5
Green TCS_2 LB 5
Green TCS_3 LB 5
Green TCS_4 LB 5
Green TCS_5 LB 5
Green TCS_6 LB 5
Green TCS_7 LB 5
Green TCS_8 LB 5
Red TCS_1 LB 5
Red TCS_2 LB 5
Red TCS_3 LB 5
Red TCS_4 LB 5
Red TCS_5 LB 5
Red TCS_6 LB 5
Red TCS_7 LB 5
Red TCS_8 LB 5

8.26
        Fluorescence intensity measuring
        Participant： Li Shisheng
Delay of Red TCS
RFU 0h 1h 2h 3h 4h 5h
1 411 431 439 489 739 1681
2 438 489 403 471 667 1690
3 431 503 541 599 820 1756
4 384 489 456 419 833 1681
5 506 434 434 530 815 1553
6 330 364 426 481 684 1501
7 361 391 482 552 739 1482
8 395 460 426 574 775 1641

Green TCS
RFU 0h 1h 2h 3h 4h 5h
1 411 516 736 917 1680 4200
2 438 554 715 907 1752 4190
3 431 534 723 923 1712 4210
4 384 702 805 839 1692 4148
5 506 590 687 878 1563 4045
6 330 577 639 891 1580 3968
7 361 588 617 860 1559 3881
8 395 569 692 992 1541 3849

8.27

        PCR
        Participant: Shisheng Li
PCR V/μL Steps Temperature/μL Time/s Cycle
super fidelity DNA polymerase 1 Pre Denature 95 180 /
tempalte 0.5 Denature 95 15 30
2X buffer 25 Annealing 55 15 30
primer-forward(10μM) 2 Extension 72 80 30
primer-reverse(10μM) 2 Final Extension 72 300 /
dNTP(10mM) 1 storage 4 / /
ddH2O 18.5 / / / /
        Template: broken Ptet-cph8 (BBa_K1886006)

8.28
    Validate the logic gate's response time. For details, see our protocol in the experiment part.
        Participant：Zhanyu Wang, Han Wan

8.30
        Verifying the double oscillation circuit
        Participant：Jinshi Ran ,Hongya Zhu
        • We co-transform the MG1655 strain with pTD103 aiiA-luxS、pTD103 luxI-sfGFP and pSB1C3 plsr-YFP.
        • We screened the positive colony and inoculated it in 50ml LB with antibiotic 100μg/ml ampicillin (Amp) and 50 mg/ml kanamycin (Kan)and Chloromycetin(C). We spun it down and concentrated it in 5ml LB when the bacteria solution reached an A600nm of 0.05~0.1.
        • We inoculated it with a 1:1000 dilution in 100ml fresh media. When the bacteria solution reached A600nm 0.2~0.3, we sampled in every 10 min and measured the fluorescence intensity.


September

9.2
        Transformation
        Participant: Shisheng Li
PCR V/μL Steps Temperature/μL Time/s Cycle
super fidelity DNA polymerase 1 Pre Denature 95 180 /
tempalte 0.5 Denature 95 15 30
2X buffer 25 Annealing 55 15 30
primer-forward(10μM) 2 Extension 72 80 30
primer-reverse(10μM) 2 Final Extension 72 300 /
dNTP(10mM) 1 storage 4 / /
ddH2O 18.5 / / / /
        Template: promoter-ccas

9.4
        Transformation
        Participant: Shisheng Li
PCR V/μL Steps Temperature/μL Time/s Cycle
super fidelity DNA polymerase 1 Pre Denature 95 180 /
tempalte 0.5 Denature 95 15 30
2X buffer 25 Annealing 55 15 30
primer-forward(10μM) 2 Extension 72 80 30
primer-reverse(10μM) 2 Final Extension 72 300 /
dNTP(10mM) 1 storage 4 / /
ddH2O 18.5 / / / /
        p-sfGFP-ccaR(BBa_K1886003)

9.7
        Transformation
        Participant： Shisheng Li
PCR V/μL Steps Temperature/μL Time/s Cycle
super fidelity DNA polymerase 1 Pre Denature 95 180 /
tempalte 0.5 Denature 95 15 30
2X buffer 25 Annealing 55 15 30
primer-forward(10μM) 2 Extension 72 80 30
primer-reverse(10μM) 2 Final Extension 72 300 /
dNTP(10mM) 1 storage 4 / /
ddH2O 18.5 / / / /
        Template: p-HO1-PCYA(BBa_K1886005)

9.9
        Validate the plasmid combining logic gate and red light-induced system: PompC+Plac (3600bp)
        Participant: Zhanyu Wang, Han Wan

9.17
        Validate the plasmid combining logic gate and green light-induced system: PcpcG2+Plac (3700bp)
        Participant：Zhanyu Wang, Han Wan



October

10.10
        PCR
        Participant: Shisheng Li
PCR V/μL Steps Temperature/μL Time/s Cycle
super fidelity DNA polymerase 1 Pre Denature 95 180 /
tempalte 0.5 Denature 95 15 30
2X buffer 25 Annealing 55 15 30
primer-forward(10μM) 2 Extension 72 80 30
primer-reverse(10μM) 2 Final Extension 72 300 /
dNTP(10mM) 1 storage 4 / /
ddH2O 18.5 / / / /
        Pompc_CL_p-lamda_sfGFP(BBa_K1886018)
        Ho1_pcyA_Pompc_CL_Plamda_sfGFP(BBa_K1886019)

Notebook for dry lab

5.19

        Started to design the circuit board for the “cipher machine” used in human practice. (Kang)

5.22

        Received the PCB from the factory, started to welding. (Ma)
        Decided to make a computer game that relate to our project.

5.27

        The PCB has some problems, we were working on with through it.

5.30

        Finished the computer game, it looks great! (Li)

6.7

        Started to design the microfluidic device. (Fu)
The electric cipher machine failed to work, Kang made a new PCB.

6.13

        Sent the blueprint of our microfluidic device to the factory.
Started to build our modeling.

6.22

        New PCB arrived, and it worked well. (Ma)

7.13

        Started to edit our wiki.

7.29

        The model for our light control system was finished, the curve is pretty good but not perfect, and we were trying to update the parameters.

8.4

        We compared the results between the modeling and the experiment. The curve is a little bit flat than we expected.

8.29

        The microfluidic device has arrived. The wet lab used it for their experiment.

9.3

        Started to build oscillation model. (Fu)

9.17

        The wiki style has been settled.

9.28

        We finished the oscillation model, but the result didn’t converge.

10.7

        After we changed some of the parameters, the result of our oscillation model finally became reasonable.

10.14

        Most of our content are finished, started to fulfill the wiki.

Contact Us
Room 413,Biology lab center, Zijingang Campus
Zhejiang University, YuHangTang Road NO.866
Hangzhou, China
iGEM ZJU-China 2016 Team
igem_zjuchina_2016@outlook.com