iGEM 2017 — Team SiCAU-China

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# Team:SiCAU-China/Notebook

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#### This page listed journals and important protocols of our project.

# Month 1（Oct 2016）

We recruited our new team members. We were busy brainstorming our project and preparing for iGEM competition. Everyone searched online and read articles for reading discussion.

# Month 2（Nov 2016）

We selected two projects for intensive study. One was “directed evolution of proteins”; the other was “action of antimicrobial peptides”. After we divided our team members into two groups and each group was responsible for one of the projects. We searched online and read many articles related to our projects, and perfected design.

# Month 3（Dec 2016）

We did a pre experiment about directed evolution of proteins, but the result was not very well. After discussion, we decided to give up the project of directed evolution of proteins, which lacked sufficient experimental conditions.

# Month 4（Jan 2017）

Focusing on tumor - targeting therapies, we wanted to construct a positive bacterium for targeting drug carriers to increase the efficiency of tumor-targeted therapy. We perfected project design and made a plan about the procedures of all the experiments.

# Month 5（Feb 2017）

We did a pre experiment about the construct of a positive bacterium, and the results showed that the experimental project is not feasible, after a series of discussion and analyses; we changed the direction of the project and turned it into exploiting Quorum-Sensing.

# Month 6（Mar 2017）

We confirmed our project. A majority of the team members wanted to be focused on detecting antibiotics. Then we searched online and read many articles related to our projects. We also consulted many experts in detecting antibiotics and discussed our projects with instructors. We also made a plan about the procedures of all the experiments.

# Month 7（Apr 2017）

We divided our team members into several groups and each group was responsible for one part of the competition. We were busy with kit construction and part construction.

# Month 8（May 2017）

We went on with part construction and lab work. We were also contacting different authorities and scheduled for community activities. We even designed questionnaires to collect public opinion about detecting antibiotics and our project.

# Month 9（June 2017）

As a result of the experimental results and other factors, we began to replace the carrier. The skeleton plasmid transformed from pSB1C3 to pSB4A5.

# Month 10（Jul 2017）

We finished a part of building jobs and continued to build other circuits.

# Month 11（Aug 2017）

We successfully constructed all our parts and expressed GFP. We began to test the fluorescence production efficiency of detecting system. Meanwhile, we did some human practices such as community propagation, interviews and collaborations with other iGEMers!

# Month12（Sep 2017）

We were busy with project optimization, wiki construction, logo design and visa application.

# Month 13（Oct 2017）

Everyone was busy with the project, from lab work, data analysis, writing staff, modeling to human practices.

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# Team:SiCAU-China/Experiments

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#### [Making Competent Cells](#mcc)

#### [PCR](#pcr)

#### [Miniprep](#min)

#### [Electrophoresis](#ep)

#### [Restriction Enzyme Digestion](#red)

#### [Ligation](#ligation)

#### [Transformation](#transformation)

#### [Fluorometric measurement](#fm)

#### [HPLC](#hplc)

![](https://static.igem.org/mediawiki/2017/2/23/T-SICAU-protocol_title.jpg)

# ![](https://static.igem.org/mediawiki/2017/6/6f/T-SICAU-Fire_paint1.jpg) Making Competent Cells

| Step | Method |
| --- | --- |
| 1 | Streak E.coli cells on an LB plate. |
| 2 | Allow cells to grow at 37°C overnight. |
| 3 | Place one colony in 10 mL LB media(+antibiotic selection if necessary), grow overnight at 37°C. |
| 4 | Take 2 ml LB media and save for blank. Transfer 500 µL overnight culture into 50 mL LB media in 250 mL flask. |
| 5 | Allow cell to grow at 37°C (200 rpm), until OD600= 0.5 (~2-3 hours). |
| 6 | Place cells on ice for 30 minutes. |
| 7 | Transfer cells to a centrifuge tube (50 mL), and centrifuge cells at 4°C for 10 minutes at 4,000×g. |
| 8 | Pour off media and resuspend cells in 12 mL of cold TB. |
| 9 | Centrifuge cells at 4°C for 10 minutes at 4,000×g. |
| 10 | Pour supernatant and resuspend cells (by pipetting) in 4 mL of cold TB and 280 µL of DMSO. Transfer 100 µL to 1.5 mL tube. |
| 11 | Ependorff tubes placed on ice. Freeze the cells in liquid nitrogen. Cells stored at -80°C can be used for transformation for up to ~6 months. |

# ![](https://static.igem.org/mediawiki/2017/2/28/T-SICAU-fire_paint2.jpg) PCR

1. Mix the following:

| Name | Volume |
| --- | --- |
| PrimeSTAR® Max DNA Polymerase | 10µL |
| Template DNA | 1µL |
| Primer Forward (10 µM) | 1 µL |
| Primer Reverse (10 µM) | 1µL |
| ddH2O | up to 20µL |
| Total | 20 µL |

2. Put samples into Thermal Cyclers and run the following steps:

| PreDenature | Denature | Anealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 98 °C | 98 °C | Tm-5 °C |72 °C -- |
| 2 min | 30 sec | 30 sec | 15 sec /kb | 30 cycles |

3. Agarose Gel Electrophoresis for confirmation.

# ![](https://static.igem.org/mediawiki/2017/2/20/T-SICAU-fire_paint3.jpg) Miniprep

| Step | Method |
| --- | --- |
| 1 | Pellet 1-4ml of overnight culture by centrifugation at 12,000×g for 1 minute. Discard the supertanant completely. |
| 2 | Add 250ul of SolutionⅠto the pellet to resuspend bacteria cells. |
| 3 | Add 250ul of SolutionⅡ, mix gently by inverting the tube 7-8 times until the solution becomes clear. The time should be no longer than 5 minutes. |
| 4 | Add 350ul of SolutionⅢ, mix gently by inverting the tube 7-8 times. |
| 5 | Centrifuge at 12,000rpm for 10 minutes. |
| 6 | Place spin column into a 1.5ml collection tube. Transfer supernatant in the step above to the column. Centrifuge at 12,000rpm for 1 minute. Discard the filtrate from the 2ml microfuge tube. |
| 7 | Return the column to the 2ml microfuge tube and add 600ul of Buffer PW. Centrifuge at 12,000×g for 1 minute. Discard the filtrate from the 2ml microfuge tube. |
| 8 | Return the column to the 2ml microfuge tube and add 600ul of Buffer PW. Centrifuge at 12,000×g for 1 minute. Discard the filtrate from the 2ml microfuge tube. |
| 9 | Place the column back into the 2ml microfuge tube. Centrifuge at 12,000×g for 2 minute. Discard the filtrate and the 2ml microfuge tube. |
| 10 | Place the spin column onto dry block heater for 8 minute. |
| 11 | Transfer the column into a clean 1.5ml microfuge tube .Add 60-80ul of Eluent or deionized water to the center of the membrane to elute the DNA. Let it stand for 1 minute at room temperature. Centrifuge at 12,000×g for 1 minute. Repeat this operation two times. Note: Pre-warm the Eluent or deionized water at 50℃ will generally improve elution efficiency. |

# ![](https://static.igem.org/mediawiki/2017/d/d8/T-SICAU-fire_paint4.jpg) Electrophoresis

|  |  |
| --- | --- |
| 1. Agalose Gel casting | 1) Measure out the appropriate mass of agarose into glass bottle with the appropriate volume of TAE buffer.  2) Microwave until the agarose is fully melted.  3) Pour the agarose solution into the gelbox and let it cool for about 30 minutes, until the gel is solid.  4) Remove comb. |
| 2.Running agalose gel | 1) Load 5 μL prepared 1kbp ladder.  2) Mix DNA solution with loading buffer (5x).  3) Load it into agalose gel.  4) Run the gel at ~110 volts for 30 minutes. |
| 3.Visualizing agarose gels | 1) Remove gel from gel box.  2) Soak the gel in ethidium bromide solution.  3) Place the gel in Trans-Illuminator and turn on UV light after make sure the door closing.  4) Print the picture.  5) Remove gel and throw in trash. |

# ![](https://static.igem.org/mediawiki/2017/1/1d/T-SICAU-fire_paint5.jpg) Restriction Enzyme Digestion

1. Mix the following:

| Name | Volume |
| --- | --- |
| Sample DNA | 4 µg |
| Restriction Enzyme (EcoRI, XbaI, SpeI, PstI) | 0.5µL |
| 10x Buffer | 1 µL |
| ddH2O | up to 10 µL |
| total | 10 µL |

2. Let stand for 1 hour at 37 °C.

# ![](https://static.igem.org/mediawiki/2017/f/f7/T-SICAU-fire_paint6.jpg) Ligation

|  |  |
| --- | --- |
| 1 | Mix the vector DNA and the insert DNA (the vector and the insert at 1 : 5-10). |
| 2 | Add 1ul Exnase® II Ligase and 2ul CE II Buffer(5x). |
| 3 | Incubate at 16 °C for 1 hour (or at 4 °C for overnight). |

# ![](https://static.igem.org/mediawiki/2017/e/e4/T-SICAU-fire_paint7.jpg)Transformation

|  |  |
| --- | --- |
| 1 | Add 2ul DNA to 150ul chemically conpetent cells on ice (set negative control by using chemically competent E.coli cells without plasmids). |
| 2 | Incubate on ice for 30 minutes. |
| 3 | Heat shock at 42℃ for exactly 90 seconds. |
| 4 | Place samples back on ice for 2 minutes. |
| 5 | Operating in the clean bench, add 900ul of LB broth per tube. |
| 6 | Incubate at 37℃ for 60 minutes, shaking. |
| 7 | Centrifuge at 4000rpm for 1 minute. |
| 8 | Operating in the clean bench, discard the supertanant (about 700ul) and resuspend bacteria cells. |
| 9 | Use the inoculating loop to load bacteria liquid then streak on the LB plate (+antibiotic selection if necessary). |
| 10 | Place plates upside down and incubate at 37℃ overnight. |

# ![](https://static.igem.org/mediawiki/2017/d/df/T-SICAU-fire_paint8.jpg) Fluorometric measurement

1. Add 2ml of the sample to quartz cell.
2. Setting measurement parameter:

|  |  |
| --- | --- |
| EX wavelength | 490nm |
| EM start wavelength | 450nm |
| EM end wavelength | 550nm |
| Scan speed | 1200nm/min |
| EX slit | 2.5nm |
| EM slit | 2.5nm |
| PMT voltage | 900V |

3. Measure by Fluorospectro photometer model: F-4600 FL Spectrophotometer

# ![](https://static.igem.org/mediawiki/2017/3/38/T-SICAU-fire_paint9.jpg) HPLC（High Performance Liquid Chromatograpy）

Inject 10μL of the sample into column.HPLC condition will show below：

|  |  |
| --- | --- |
| HPLC | Agilent 1290 Infinity LC |
| Column | ZORBAX Eclipse® XDB-C18 |
| Total Flow | 1mL/min |
| Pump A | Pump B=H2O |
| temperature | 25℃ |
| Mobile phase | Pump A:Pump B=H2O:MeOH=0.6:0.4 |

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# Team:SiCAU-China/Results

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# ![](https://static.igem.org/mediawiki/2017/6/6f/T-SICAU-Fire_paint1.jpg) Detect the E.coli BL21-4A5-R-IG fluorescence intensity

We acquired the strains E.coli BL21-4A5-R-IG with recombinant plasmid vector pSB4A5-R-IG, according to the initial experimental methods.

     The recombinant colonies was transferred into LB culture medium with ampicillin , and shaked overnight at 37℃, then took the medium for fluorescence detection. We found the fluorescence signal was weak (Figure 1) and cannot be detected under the condition of its background expression as we expect. Thus it is speculated that the phenomenon may be caused by the low effective expression of the LuxI protein, in which cases there is not enough AHL.

![](https://static.igem.org/mediawiki/2017/e/ef/T-SICAU-result_figure1.jpg)

Figure 1: Regard the fluorescence of Ecoli BL21 as “1”, the relative fluorescnce of E.coli BL21-4A5-R-IG after culturing 20h.

![](https://static.igem.org/mediawiki/2017/6/6b/T-SiCAU-result-fig1.jpg)

Figure 2: Left:E.coli BL21-4A5-R-IG; Left:E.coli BL21

To test our hypothesis, we next verified the module-LuxI. The fluorescence intensity significantly increased after adding different concentrations of AHL (Figure 3). Therefore, the system was probably activated by adding AHL which indicated that there was a problem with the LuxI module. By analyzing the LuxI gene sequence, we found out there is an LVA tag in LuxI module, which was cloned from iGEM part (BBa\_C0061). This tag will accelerate the degradation rate of its corresponding proteins, so that it can prevent the LuxI protein from playing a stable role in cell. It may, therefore, be concluded that the AHL signal was too weak to activate the positive feedback system.

![](https://static.igem.org/mediawiki/2017/9/96/T-SICAU-result_figure2.jpg)

Figure 3：Regard the fluorescence of E.coli BL21-4A5-R-IG without AHL as “1”, the fluorescence of E.coli BL21-4A5-R-IG added different concentrations of AHL after being cultured 20h.

# ![](https://static.igem.org/mediawiki/2017/2/28/T-SICAU-fire_paint2.jpg) Delete the LVA tag of LuxI

After fermenting E.coli BL21-4A5-R-IG-△LVA and adding AHL at the same time, we measured fluorescence values comparing with the control group. The fluorescence value of test group increased rapidly (Figure 4).
     Our system was activated that indicated the positive feedback was effective. However, after 20 hours, the AHL-free control group was still up to xx, in other words, the system background expression could also make the system to be activated and enable the positive feedback system to be constructed successfully.

![](https://static.igem.org/mediawiki/2017/a/a4/T-SICAU-result_figure3.jpg)

Figure 4: The fluorescence/OD600 of E.coli BL21-4A5-R-IG-△LVA after adding AHL 6h and 20h. After being cultured 6h, the fluorescence of E.coli BL21-4A5-R-IG-△LVA added 10^-8 AHL was about twice as without being added AHL. While the fluorescence was almost indifferent between the two groups after 20h.

![](https://static.igem.org/mediawiki/2017/1/14/L-SiCAU-result-fig2.png)

Figure 5: Different bacteria after 20h culturing, from left to right: E.coli BL21-4A5-R-IG(△LVA); E.coli BL21-4A5-R-IG adding AHL(10^-8M); E.coli BL21-4A5-R-IG with no AHL; E.coli BL21

# ![](https://static.igem.org/mediawiki/2017/2/20/T-SICAU-fire_paint3.jpg) E.coli BL21-4A5-R-IG-△LVA add lac control area

In order to avoid the impact of system detection limits and the influences of the prolonged cell growth period (See [modeling](https://2017.igem.org/Team%3ASiCAU-China/Model) for details), we added lac operon to the positive feedback system expecting the positive feedback system to be activated when IPTG was added into the culture medium in the stationary period of E.coli.
     With adding IPTG, the results showed that the positive feedback was normal; without IPTG, the positive feedback was closed (Figure 6). This indicated that the PLux/Lac switch functions well.
     Then we added IPTG to the medium in the cell stationary phase. After 12h induction, no positive feedback was found, which means the system didn’t work normally.
And then AHL was added in the stationary phase, in that case the positive feedback system still didn’t work . Through reading lots of literature, we speculated that model-LuxR may not work in the stable period, leading to the failure of activating positive feedback system [1]. (If you want to read more details, you can click here to see our [contribution](https://2017.igem.org/Team%3ASiCAU-China/Contribution))
     So we would rather choose logarithmic growth period than stationary phase to detect the input signal.

![](https://static.igem.org/mediawiki/2017/e/e2/T-SICAU-result_figure4.jpg)

Figure 6:The fluorescence/OD600 of E.coli BL21-4A5-R-IG-△LVA-lac(+lacI) after being culturd 20h; IPTG(-)without adding IPTG; IPTG（L+）adding IPTG in logarithmic growth period;IPTG(S+) adding IPTG in stationary phase.

# ![](https://static.igem.org/mediawiki/2017/d/d8/T-SICAU-fire_paint4.jpg) Determine the amplification effect of the system

After adding a series of concentrations for AHL, we measured fluorescence of E.coli BL21-4A5-RG and E.coli BL21-4A5-R-IG-△LVA. The results showed that the measurement accuracy of E.coli BL21-4A5-R-IG-△LVA could reach the concentration of 10^-9M(Figure 7) while the one of E.coli BL21-4A5-RG, which has no positive feedback, was 10^-8M. As for the ordinary high performance liquid chromatography, when AHL concentration accuracy reached 10^-6M (Figure 8), its detection was not very obvious. Comparing the measurement accuracy of E.coli BL21-4A5-R-IG-△LVA with that of E.coli BL21-4A5-RG, the former is approximately an order of magnitude higher than the latter. When compared with High Performance Liquid Chromatography(HLPC), the measurement accuracy of E.coli BL21-4A5-R-IG-△LVA was nearly three orders higher than that of HLPC. To achieve the accuracy of 10^-9M, you can also use HPLC-TMS, but its operation will be slightly complex[2].

![](https://static.igem.org/mediawiki/2017/7/74/T-SICAU-result_figure5.jpg)

Figure 7: The fluorescence/OD600 of E.coli BL21-4A5-R-IG-△LVA added different gradient concentrations of AHL and measured at different time

![](https://static.igem.org/mediawiki/2017/f/f7/T-SICAU-result_figure6%281%29.jpg)![](https://static.igem.org/mediawiki/2017/8/84/T-SICAU-result_figure6%282%29.jpg)

Figure 8:The left one is 10^-2 AHL;The right one is 10^-6 AHL.Both are measured by HLPC.

# ![](https://static.igem.org/mediawiki/2017/1/1d/T-SICAU-fire_paint5.jpg) Control the background expression of AHL

A high background expression can affect the detection limits of the system. Fortunately, AiiA hydrolytic enzymes can hydrolyze the AHL protein while LVA tag accelerates degradation of LuxI, thereby reducing the generation of AHL. Both of these ways are able to control AHL background expression. However, the effect of LVA tag will be strengthen with the enlargement of positive feedback system. The reason is that LVA tag exists on model LuxI. With the expression of LuxI, the total number of intracellular tags will increase accordingly, which may lead to a too strong control ability on the positive feedback effect, during the stationary period. According to the Michaelis-Menten equation, when the AHL is enough, the degradation rate of AHL is constant. So using AiiA to control background expression would not have a significant effect on the positive feedback. Thoughtfully, we chose to add AiiA for system control. When compared with E.coli BL21-4A5-R-IG-△LVA, the added AiiA will delay the threshold, but the increase rate is basically same as before(Figure 9), which was in line with our expectations. The decrease in the end point may be caused by the thing that the positive feedback effect had already stopped before cells reached the stationary period, which also confirmed our previous speculation that LuxR did not work stationary period.

![](https://static.igem.org/mediawiki/2017/2/2b/T-SICAU-result_figure7.jpg)

Figure 9: The fluorescence/OD600 of E.coli BL21-4A5-R-IG-△LVA-AiiA added different gradient concentrations of AHL and measured at different time.

# ![](https://static.igem.org/mediawiki/2017/f/f7/T-SICAU-fire_paint6.jpg) After accessing to tetracycline converter

We constructed E.coli BL21-1C3-RT to detect whether the converter is normal or not. It can be found that the background level was high, which means the double plasmid system may need more AiiA hydrolase to control the background expression of AHL. We intended to switch to a stronger promoter or RBS to improve AiiA expression, but due to the limited time, the improvement hasn’t been done.
     If we could solve the problem of AHL background expression ,we would isolate E.coli BL21-4A5-AiiA/1C3-IT which contains both pSB4A5-R-IG-△LVA-AiiA and pSB1C3-LuxI-TetR plasmids, then add different concentrations of Tetracycline into LB medium to detect performance of that system.

# Future work:

1. Change the positive feedback system to make sure that can work in a stationary phase
     2. Change the different intensities of the promoter or RBS to eliminate background expression

# References

[1] Zhu J. Winans SC. Auto inducer binding by the quorum sensing regulator TraR increases affinity for target promoters in vitro and decreases TraR tumover rates in whole cells. Proc.Natl.Acad. Sci. USA, 1999，96:4832-4837.
     [2] Chenchen Ma, Jie Ou, Bolin Li. Method for measuring AHLs by HPLC-TMS:CN 103063774 B[P]. 2014.

# Plasmids

| Number | Plasmid name |
| --- | --- |
| 1 | *pSB4A5-RG* |
| 2 | *pSB4A5-R-IG*(BBa\_K2311000) |
| 3 | *pSB1C3-RFP-TetR* |
| 4 | *pSB1C3- LuxI-TetR* |
| 5 | *pSB4A5-R-IG-△LVA*(BBa\_K2311002) |
| 6 | *pSB4A5-R-IG-△LVA-lac5* |
| 7 | *pSB4A5-R-IG-△LVA-AiiA*(BBa\_K2311001) |

# Strains

| Number | Strain name |
| --- | --- |
| 1 | *E.coli BL21-4A5-RG* |
| 2 | *E.coli BL21-4A5-R-IG* |
| 3 | *E.coli BL21-1C3-RT* |
| 4 | *E.coli BL21-4A5-R-IG-△LVA* |
| 5 | *E.coli BL21-4A5-R-IG-△LVA-lac* |
| 6 | *E.coli BL21-4A5-R-IG-△LVA-AiiA* |

     If you want to read more details about how to construct the plasmids and strains, you can click here to see our [protocol](https://2017.igem.org/Team%3ASiCAU-China/Experiments) and [notebook](https://2017.igem.org/Team%3ASiCAU-China/Notebook)!

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