Team:ZJU-China/Experiments
Light Control
To achieve the function of the first generation of our cipher machine, our bacteria should be sensitive enough to the light signal around specific wavelength long and output a fluorescent signal which should be strong enough to be detected. So we designed several experiments to prove the sensitivity and reliability of the two TCSs.
Firstly, we chose sfGFP as the reporter and exposed our bacteria to red or green light when culturing. After some time, we harvested all the culture and measured the fluorescent intensity .
What’s more , to make our cipher machine more complicated and hard to be cracked, we induced the oscillation circuit. But the key to couple these parts was to prove that the time our “light-sensors” need between sensing the light and output a signal was exactly shorter than the half-period of oscillation. So we tried to measure this delay time of the two TCSs. We also tried to stimulate this process by modeling.
Here we want to show our sincere appreciation to Team HZAU who had provided us with original plasmids the paper used, also to Team XJTLU who had designed the device for culturing the bacteria and measuring the fluorescent intensity.
Logic gate
In order to validate the feasibility of the logic gate part, we designed experiment from the following aspects.
1. Qualitative experiment
We set 4 experimental groups and 1 control group to validate the logic gate qualitatively. The control group is MG1655 without plasmid that contains the logic gate circuit transformation. The 4 experimental groups are MG1655 with target plasmid transformation. And the treatments are: 1) adding nothing, 2) adding IPTG, 3) adding ARA, 4) adding both IPTG and ARA.By comparing the data from these 5 groups, we can find if there is any leakage of the two promoter, and validate the circuit.
2. Quantitative experiment
We set a series of concentration gradient for ARA and IPTG from 10-7M/L to 10-3M/L. The specific setting is as follows.
3. The exploration of the response time
In order to explore the response time of the logic gate circuit, we change the time of adding ARA and IPTG. We set several experimental groups at one-hour intervals, and measure the fluorescence intensity to find the response time.
Oscillation
1. Determination of the relationship between the concentration of AHL molecule and the pluxR response strength
In order to understand and describe the state of the single period oscillation system better, we need to know the relationship between the pluxR response strength and the AHL molecular concentration. To this end, we designed the AHL concentration gradient from 10^-11 to 10^-6, and used it for the induction of bacteria that contain pLuxR-sfGFP report loop. After fully cultured, the fluorescence intensity was measured, and we can get the concentration of AHL -- fluorescence intensity diagram.
2. Detection of single period oscillation system
In order to detect single-period oscillation system composed of pTD103aiiA and pTD103luxI-sfGFP, these two plasmids were co-transfected into MG1655.The selected positive clones were cultured overnight. We inoculated them in 96-well plates in 1:1000, culture under 37°C and check the fluorescence intensity of GFP, to monitor the occurrence of oscillation.
3. Validation of the single period oscillation system in a microfluidic device
By analyzing the data, we found that, because of the increase in the amount of bacteria as well as the accumulation of AHL, the oscillation will become unstable, and the whole system will collapse, too. In order to eliminate the influence of all above, we designed a microfluidic device. Microfluidic chip can provide sufficient nutrition for the growth of the bacteria and wash away excess bacteria and AHL, so that the system can run stably. Under the fluorescence microscope, we captured pictures every 5min.
4. The construction of double period oscillation system
In order to realize the double periodic oscillation, we also need to introduce another kind of auto-inducer into the oscillation system, we chose DPD, another auto-inducer in E.coli. We connected RBS with luxS to aiiA, which allows them to co-express. LuxS catalyzes the formation of DPD, so that the concentration of DPD in theory will be a half cycle difference with the peak of AHL, we named it as pTD103aiiA-luxS. PLsr is regulated by DPD promoter, it is followed by YFP as a reporter gene, so that we can monitor the change of the concentration of DPD, we named it as pTD103YFP. pTD103aiiA-luxS, pTD103luxI-sfGFP and pTD103YFP three plasmids constitute a double cycle system, sfGFP and YFP as reporter gene. We hope to observe the green and yellow light stable appear alternately, and each cycle length is fixed.
5. Detection of double periodic oscillation system
As the method we used for the detection of single period oscillation, we put the treated bacteria solutio into the microfluidic device, and inject the fresh LB with proper flow rate, in order to stabilize the system. Under the fluorescence microscope, we captured pictures every 5min.
Documentation
PART A Brainstorming and Information Gathering
5/1-6/28
1 Brainstorming for ideas
5/1-5/31
Voting and deciding on our project: ENIGMA
5/31
2 Searching for relevant information and optimizing the idea
6/1-6/18
3 Experiments preparation
6/18-6/30
4 Getting advice on the project
6/1-now
PART B Experiments
6/29-10/10
Module 1 Photo-related control system
1 Plasmid construction (Green Light & Red light Plasmids)
6/30-7/20
2 Function verification
7/20-8/30
3 Modeling
7/15-8/30
4 Coupling with other modules
Module 2 Oscillation system
1 Plasmid construction (Single & Double period)
6/30-7/15
2 Device: Micro-fluidics
7/2-7/20
Design
7/2-7/10
Production
7/10-7/20
3 Demonstration of single period oscillation
In a tube
7/16-7/25
In Micro-fluidics
7/16-7/25
4 Demonstration of double period oscillation
8/5-8/30
5 Modeling
7/15-8/30
6 Coupling with other modules
Module 3 Logic Gates
1 Plasmid construction (Improvements and Adjustments)
6/30-7/28
2 Function verification (Previous parts and new parts)
7/20-8/30
3 Modeling
7/15-8/30
4 Coupling with other modules
Coupling 1 Photo-related control system & Logic gates (Cipher Machine of the First Generation)
1 Plasmid construction (Basing on existing plasmids)
9/1-9/10
2 Demonstration
9/10-9/25
3 Modeling
9/8-9/25
Coupling 2 All three Modules (the ENIGMA machine)
1 Plasmid construction (Basing on existing plasmids)
9/20-10/3
2 Demonstration
10.4-Now
PART C Human Practice
1 Synbiobeta in Shanghai
6/24
2 Tianjin Exchange Conference
7/2
3 Six-school Exchange Conference in Zhejiang University
7/7-7/8
4 STEM Festival in Suzhou
7/16-7/17
5 Exchange Conference with HZAU
7/22
6 Wuhan Science & Technology Museum
8/31
7 Central China iGEM Consortium(CCiC )
9/2-9/4
8 One Youth Talk
9/25
PART D Preparation for Giant Jamboree
Wiki Designing and writing
9/15-10/17
Poster Designing
10/2-10/15
Presentation preparation (PowerPoint, draft of the speech)
10/2-10/25
Protocol
Protocol for chemical inducible expression of GFP
Materials:
1、4 groups of induce solution with a concentration gradient of 10^-7, 10^-5, 10^-3, 10^-2;
2、 Overnight bacterial culture or bacterial colonies;
3、Phosphate Buffered Solution (PBS).
Procedure:
1. Add 20 μ l of the overnight bacterial culture or pick a colony to 5 ml of LB antibiotic medium, Incubate at 37 degree in a shaker till the OD600 value reaches 0.4-0.6.
2. Add 0.5 mL of the fresh bacterial 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, 10^-2.
3. Place the induction system at 37 degree for 2 hours.
4. Pellet bacterial cells by 4 min centrifugation at 4000 rpm, discard the supernatant.
5. Resuspend the pelleted cells in 500 μ l of PBS.
6. Transfer 100 uL of bacterial resuspention into each well of 96-well plate to test the expression of GFP by flow cytometry or Microplate Reader.
1. Late in the day, start a 37 °C, shaking overnight culture from a −80 °C stock in a tube containing 3 mL LB medium and the appropriate antibiotics (50 µg/mL kanamycin, 100 µg/mL spectinomycin and 34 µg/mL chloramphenicol for the CcaS-CcaR system, and the same antibiotics for the Cph8-OmpR system).
2. After the overnight culture has grown for 10–12 h, prepare 200 mL LB medium. Add appropriate antibiotics to medium. Shake/stir the container to ensure the antibiotics are mixed well in the medium.
3. Measure the OD 600 of the overnight culture.
4. Dilute the overnight culture into the LB + antibiotics, bringing the OD 600 to 0.0001. Shake/stir the container to ensure the cells are mixed well in the medium.
5. Distribute 10 mL of inoculated medium into each of15ml tubes.
6. Place tubes in the shaker and grow at 37 °C with shaking at180r.p.m. for 8 h. Expose the tubes under light of specific wavelength.
7. After 8 h of growth, harvest all test tubes by immediately transferring them into an ice-water bath. Wait 10 min for the cultures to equilibrate to the cold temperature and for gene expression to stop.
8. Approximately 1.5 h before stopping the experimental cultures, begin preparing a solution of phosphate-buffered saline(PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 2 mMKH2PO4 , pH to 7.4) + 500 µg/mL of the transcription inhibitorrifampicin .Rif dissolves slowly, so allow at least 45 min of stirring. Also at this time, begin preparing a 37 °C water bath.
9. Filter the dissolved solution of PBS + Rif through a0.22-µm 20-mL syringe filter.
10.6000r.p.mcentrifuge the culture for 3 minutes and refloat with PBS+Rif
11. 37 °C water bath for 1 h.
12. Transfer the samples back into ice-water bath.
13. Wait 15 min, and then begin measuring each tube on a multiple plate reader.
Protocol for measuring and modeling of promoter pluxR
Materials
1.Mutated plasmid Ptd103sfGFP
2.Competent cell of MG1655 strain of E·coil
3.LB media
4.Phosphate Buffered Solution (PBS).
5.Kanamycin
6.N-Hexanoyl-L-homoserine lactone
Procedure:
1.mutate pTD103luxI-sfGFP with knocking gene luxI out with PCR. We named the new plasmid pTD103sfGFP.
2.Transform MG1655 strain of E·coil with pTD103sfGFP, inoculated positive colony in 5ml LB with antibiotic 100μg/ml kanamycin and cultured it over night.
3.Inoculate 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,. Culture it in 37℃ for 8h.
4.Spin it down and concentrate in PBS (pH=7.2), measure the fluorescence intensity.
Verifying the single oscillation circuit
Materials：
1.Plasmids pTD103luxI-sfGFP and pTD103aiiA-luxS、pSB1C3 plsr-YFP
2.Competent cell of MG1655 strain of E·coil
3.M9 media
4.Kanamycin、Ampicillin、Chloromycetin
Procedure：
1.Co-transform the MG1655 strain with pTD103aiiA and pTD103luxI-sfGFP.
2.Screen the positive colony and inoculate in 50ml M9 media with antibiotic 100μg/ml ampicillin (Amp) and 50 mgml21 kanamycin (Kan).
3.spin it down and concentrate it in 5ml M9 media when the bacteria solution reach an A600nm of 0.05~0.1.
4.inoculate re-suspension with a 1:1000 dilution in 100ml fresh M9 media, when A600nm 0.1, add to 96 wells plate and measure the fluorescence intencity in 37℃.
Protocol for verifying the single oscillation circuit in microfluidics device
Materials:
1.Plasmids pTD103luxI-sfGFP and pTD103aiiA
2.Competent cell of MG1655 strain of E·coil
3.LB media
4.Kanamycin、Ampicillin
5.Tween 20
Procedure:
1.Co-transform the MG1655 strain with pTD103aiiA and pTD103luxI-sfGFP.
2.Screen the positive colony and inoculate in 50ml LB with antibiotic 100μg/ml ampicillin (Amp) and 50 mg/ml kanamycin (Kan).
3.Spin it down and concentrate it in 5ml of fresh media with surfactant concentration of 0.075% Tween20 when the bacteria solution reach an A600nm of 0.05~0.1.
4.Load the sample from the cell port while keeping the media port at sufficiently higher pressure than the waste port below to prevent contamination. 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.
5.Take pictures of microfluidics in fluorescence microscope every 5 mins
Verifying the double oscillation circuit
Materials:
1.Plasmids pTD103luxI-sfGFP and pTD103aiiA-luxS、pSB1C3 plsr-YFP
2.Competent cell of MG1655 strain of E·coil
3.M9 media
4.Kanamycin、Ampicillin、Chloromycetin
Procedure:
1.Co-transform the MG1655 strain with pTD103 aiiA-luxS、pTD103 luxI-sfGFP and pSB1C3 plsr-YFP.
2.Screene the positive colony and inoculate it in 50ml M9 media with antibiotic 100μg/ml ampicillin (Amp) and 50 mg/ml kanamycin (Kan)and Chloromycetin(C).
3.Spin it down and concentrate it in 5ml M9 media when the bacteria solution reach an A600nm of 0.05~0.1.
4.Inoculate re-suspension with a 1:1000 dilution in 100ml fresh M9 media. When the bacteria solution reache A600nm 0.1， add to 96 wells plate and measured the fluorescence intensity every 3min.
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
igem_zjuchina_2016@outlook.com
