Difference between revisions of "Team:ZJU-China/Notebook"

Latest revision as of 11:28, 19 October 2016

Notebook

June

6.15

        Participant: Zhanyu Wang, Han Wan
        1.Get all the basic parts from Kit plate
        supD-tRNA ,T7RNAP &PT7

Gene of interest	Vector	Ice	42°C	Ice	37°C+LB	Plate
Transformation
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

Gene of interest	Vector	Ice	42°C	Ice	37°C+LB	Plate
Transformation
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

Name	Medium	Volume/ml
Liquid Culture
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

Name	260/280	C ng/μl
Miniprep
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

Gene of interest	Vector	Ice	42°C	Ice	37°C+LB	Plate
Transformation
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

Name	Medium	Volume/ml
Liquid Culture
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

Name	260/280	C ng/μl
Miniprep
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

Name	Medium	Volume/ml
Liquid Culture
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

Gene of interest	Vector	Ice	42°C	Ice	37°C+LB	Plate
Transformation
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

Name	Medium	Volume/ml
Liquid Culture
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

Gene of interest	Vector	Ice	42°C	Ice	37°C+LB	Plate
Co-Transformation
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

Name	Medium	Volume/ml
Liquid Culture
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

RFU	Control	Dark/th>	green light	red light
Green TCS
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

RFU	Control	Dark/th>	red light
Red light TCS
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

Name	Medium	Volume/ml
Liquid Culture
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

RFU	0h	1h	2h	3h	4h	5h
Delay of Red TCS
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

RFU	0h	1h	2h	3h	4h	5h
Green TCS
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

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