iGEM 2019 — Team BIT-China

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# Team:BIT-China/Notebook

![](https://static.igem.org/mediawiki/2019/8/8a/T--BIT-China--arrow.png)

Experiments

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# Team:BIT-China/Experiments

![](https://static.igem.org/mediawiki/2019/8/8a/T--BIT-China--arrow.png)

Background

a

# Experiment

![](https://static.igem.org/mediawiki/2019/1/15/T--BIT-China--resource_experiment_1.png)

#### The production of seed fermentation broth

The sequenced strains were transferred to petri dishes and activated. Then we transferred the activated strain to a single colony in LB medium containing 0.1% Amp.

Shake flask fermentation:
Add 20ml LB medium containing 0.1%Amp into 50ml conical flask. Measure seed fermentation broth concentration with an ultraviolet spectrophotometer. Transfer the seed fermentation broth to 20 mL LB medium (0.1%Amp) when the OD600 is 0.05 Abs. Culture in shaking table for 12h, sampling every 2 hours.

Fluorescence values and OD600 data at the excitation wavelength of 480nm and emission wavelength of 517 nm were measured and the results were as follows：

![](https://static.igem.org/mediawiki/2019/9/92/T--BIT-China--resource_experiment_2.png)

#### Result analysis

Because the strain transferred from the seed fermentation broth to the conical flask turned on the QS system and the green fluorescence was expressed, we took the initial fluorescence value after culture for four hours. As shown in figure 1, with the passage of fermentation time, the green fluorescence value gradually increased, and the QS system of the strain was turned on. However, after two hours of opening, excessive quorum sensing signals inhibited the promoter pLUX, leading to the gradual shutdown of the QS system. The low expression of the strong promoter J23102 in the QS strain of green fluorescent protein indirectly indicates that quorum sensing signal molecules have negative effects on the QS system. With the decrease of quorum sensing semaphore, pLUX can be turned on normally to realize the expression of green fluorescent protein. After 10 hours of culture, fluorescence was no longer significantly increased or decreased. Maybe the nutrient content in the culture medium was too low, which affected the growth of escherichia coli, and the population density would not increase too much. The 12h growth curve is shown in figure 2.

![](https://static.igem.org/mediawiki/2019/7/73/T--BIT-China--resource_experiment_3.png)

Prove that the σF and σB provided by the literature are orthogonal to the σ factor of the basal cells.

1. PET28a-pBAD-σB-K1150012-PσB-mcherry-B0015

(1) Used PCR to amplify pBAD, σB, K1150012, mcherry, B0015, pBAD, σF and K1150012.

![](https://static.igem.org/mediawiki/2019/2/20/T--BIT-China--resource_experiment_4.png)

(2) Used OE-PCR to connect pBAD-σB, K1150012-pσB and mcherry-B0015, pBAD-σB-K1150012, pBAD-σF-K1150012 and pσB-mcherry-B0015.

![](https://static.igem.org/mediawiki/2019/d/d7/T--BIT-China--resource_experiment_5.png)

2. Construction of feedback gene line PET28a-pLUXI-σF-K1150012-PσF-mcherry-B0015

![](https://static.igem.org/mediawiki/2019/d/d6/T--BIT-China--resource_experiment_6.png)

(1) Used PCR to amplify σF, (F)K1150012, (F)mcherry, (F)B0015, σB, (B)K1150012, (B)mcherry.

(2) PET28a-pLUXI-σB-K1150012-PσB-mcherry-B0015.

![](https://static.igem.org/mediawiki/2019/6/60/T--BIT-China--resource_experiment_7.png)

(3) Used OE-PCR to connect K1150012, PσF and mcherry, K1150012, PσB and mcherry.

![](https://static.igem.org/mediawiki/2019/f/f4/T--BIT-China--resource_experiment_8.png)

(4) Used Gibson assembly pLux-σF-K1150012-PσF-mcherry-B0015 and vector pET28a.

![](https://static.igem.org/mediawiki/2019/c/ce/T--BIT-China--resource_experiment_9.png)

3. Verify the presence of a promoter leak in the gene line

Plasmids for pET28a-mcherry-B0015 and pET28a-σF-K1150012-PσF-mcherry-B0015 were obtained by PCR from pET28a-pLuxI-σF-K1150012-PσF-mcherry-B0015 plasmid template.

![](https://static.igem.org/mediawiki/2019/6/62/T--BIT-China--resource_experiment_10.png)
![](https://static.igem.org/mediawiki/2019/1/13/T--BIT-China--resource_experiment_11.png)
![](https://static.igem.org/mediawiki/2019/0/02/T--BIT-China--resource_experiment_12.png)

4. pUC19-σF-σB-T7RNAP-K1150012

![](https://static.igem.org/mediawiki/2019/d/dd/T--BIT-China--resource_experiment_13.png)

(1) Used OE-PCR to conect σF and σB.

![](https://static.igem.org/mediawiki/2019/2/2b/T--BIT-China--resource_experiment_14.png)

(2) Used Gibson assembly to connect σF-σB, T7RNAP, K1150012 and vector pET28a.

![](https://static.igem.org/mediawiki/2019/3/3e/T--BIT-China--resource_experiment_15.png)

5. Final gene line connection

![](https://static.igem.org/mediawiki/2019/f/fc/T--BIT-China--resource_experiment_16.png)
![](https://static.igem.org/mediawiki/2019/0/08/T--BIT-China--resource_experiment_17.png)

6. Fermentation

![](https://static.igem.org/mediawiki/2019/e/e8/T--BIT-China--resource_experiment_18.png)

The production of seed fermentation broth: The sequenced strains were transferred to petri dishes and activate, Then we transferred the activated strain to a single colony in LB medium containing 0.1% Amp.

#### Shake flask fermentation

Add 20ml LB medium containing 0.1%Amp into 50ml conical flask. Measure seed fermentation broth concentration with an ultraviolet spectrophotometer. Transfer the seed fermentation broth to 20mlLB medium (0.1%Amp), when the OD600 is 0.05Abs. Culture in shaking table for 12h, sampling every 2 hours. Fluorescence values and OD600 data at the excitation wavelength of 480 nm and emission wavelength of 517 nm were measured and the results were as follows：

![](https://static.igem.org/mediawiki/2019/f/f4/T--BIT-China--resource_experiment_19.png)

According to the description of promoter strength provided by the official website of IGEM, we conducted different strength promoter replacement experiments for QS quorum sensing circuit. We investigated the effects of different LuxR contents on QS system and the growth of bacteria. The results showed that promoter J23102 with high intensity was not sensitive to the population density of E.coli. Although J23102 can realize the opening of QS population density induction circuit, the promoter J23117 with the weakest expression intensity is more suitable for the opening of QS population density system in E.coli. At the same time, the change of different promoters indicated that LuxR would not affect the growth of e. coli to some extent.

![](https://static.igem.org/mediawiki/2019/1/19/T--BIT-China--resource_experiment_20.png)

Construction of gene expressing vector: We connected pT7, psigmaF, psigmaB and mcherry to the plasmid pACYCDute-1, then we uesd fluorescence intensity to characterize promoter intensity.

#### Experimentation

The plasmid was transformed to *E. coli* BL21, then we transferred the *E. coli* colonies that were successfully transformed into LB medium containing chloramphenicol. After 12 hours of culture, it was transferred to 40 ml LB medium containing chloramphenicol, with initial OD600 of 0.01. Samples of E.coli containing psigmaF, psigmaB were taken every two hours. After 5 hours of transfer of E.coli containing T7 promoter, 1/1000 IPTG was added, and samples were taken every 2 hours for a total of 12 hours.

The results are shown below:

![](https://static.igem.org/mediawiki/2019/8/85/T--BIT-China--resource_experiment_21.png)

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# Team:BIT-China/Results

![](https://static.igem.org/mediawiki/2019/8/8a/T--BIT-China--arrow.png)