iGEM 2023 — Team xju-china


Engineering | XJU-China
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Overview
1. Tests of salinity and base resistance of wild bacterias
2. Gene mining system
3．Construction of engineering bacteria
4 Successful mining of components

# OVERVIEW

Relying on such an engineering model, we succeeded in obtaining universal gene originals in non-model strains that can improve the salinity and base resistance of model strains.

The experiment proves that our engineering is very successful, and such an experimental idea can also be used for mining other elements, we welcome every teams who want to do this work to communicate and use this method.

I will describe the method in detail and show the success we achieved in this page.

![](https://static.igem.wiki/teams/4835/wiki/engneer/lc2.png)

# 1. Tests of salinity and base resistance of wild bacterias

We collected wild strains from different habitats, purified them and tested for salinity tolerance. Six strains with good tolerance to salinity were obtained, and the results are shown in the figure.

![](https://static.igem.wiki/teams/4835/wiki/engneer/limit.jpg)

# 2. Gene mining system

![](https://static.igem.wiki/teams/4835/wiki/engneer/eng-liucheng.png)

Northwestern University Invitation

## 2.1 Method

To identify the common genetic elements differentially expressed in transcription, translation and metabolism that were commonly altered by the six salt-tolerant bacterial strains in response to salinity stress, we used p < 0.05 and |log2FC| > 0 as screening thresholds for the differentially expressed genes and differentially expressed proteins.

## 2.2 Result

There were two up-regulated differentially expressed genes, sufB (iron-sulfur cluster scaffolding complex subunit) and mnmA (tRNA-specific 2-thiouracilase), and no down-regulated differentially expressed genes among the six strains; there was one up-regulated differentially expressed protein, purM among the six strains.

The six strains of salinity-tolerant bacteria were screened for differentially expressed genes on both the transcriptome and proteome without differentiating between up- and down-regulation, and found that the six strains had 19 differentially expressed genes, the six strains had 21 differentially expressed proteins, and the six strains had five differentially expressed genes on both the transcriptome and proteome.（details are shown in model section）

In summary, a total of 40 genes with high likelihood were screened, and finally, in combination with the metabolomic data, the six most probable universal antisaline elements expressed at transcriptional, translational, and metabolic levels by saline-tolerant bacteria of different genera in response to different saline environments were listed. The results are as follows.

![](https://static.igem.wiki/teams/4835/wiki/engneer/t5-9.png)

![](https://static.igem.wiki/teams/4835/wiki/engneer/t5-10.png)

![](https://static.igem.wiki/teams/4835/wiki/engneer/dna-result.png)

The gene we found

# 3．Construction of engineering bacteria

## 3.1 Plasmid construction

We chose T7 promoter and pet28a to construct recombinant plasmids. The pET-28a(+) vector and the target gene were double digested by Nco I and Hind III, and the gene was ligated to the vector using T4 ligase to obtain recombinant expression vectors of different sizes.

The following pictures show the recombinant plasmids we constructed and some of mappings

![](https://static.igem.wiki/teams/4835/wiki/engneer/zl-ppt1.png)

![](https://static.igem.wiki/teams/4835/wiki/engneer/zl-ppt2.png)

![](https://static.igem.wiki/teams/4835/wiki/engneer/zl6.png)

We have identified the surfB gene in five bacterial strains and have completed the detection of surfB. Here are the detailed plasmid mapping.

![](https://static.igem.wiki/teams/4835/wiki/engneer/zl1.png)

![](https://static.igem.wiki/teams/4835/wiki/engneer/zl2.png)

![](https://static.igem.wiki/teams/4835/wiki/engneer/zl3.png)

![](https://static.igem.wiki/teams/4835/wiki/engneer/zl4.png)

![](https://static.igem.wiki/teams/4835/wiki/engneer/zl5.png)

## 3.2 Plasmid verification

In order to verify whether the target gene was successfully constructed into the expression vector, the reaction system was digested at 37 ℃ for three hours, and then the results were detected by agarose gel electrophoresis (1% agarose gel (containing 0.5 μ g / mL nucleic acid dye), 90V electrophoresis for 30 minutes.

![](https://static.igem.wiki/teams/4835/wiki/engneer/maker1.png)

Results:

By observing the results of enzyme digestion, it was found that the band was consistent with the length of the target gene, which indicated that the target gene was successfully constructed into the expression vectorpET28a and could be transformed later.

## 3.3 Element effect detection

Before testing the effect of the components, we first ensured that the target gene surfB was successfully introduced into E. coli through experiments.

![](https://static.igem.wiki/teams/4835/wiki/engneer/result-maker.png)

Results:

The constructed plasmid was added to the prepared competent cells, and after treatment, in order to verify whether the plasmid was transformed into the cells, the cells with the target plasmid were cleaved and then digested with endonuclease. The results of enzyme digestion were detected by agarose gel electrophoresis.

## 3.4 protein detection

![](https://static.igem.wiki/teams/4835/wiki/engneer/protein.png)

# 4 Successful mining of components

Through the above steps, we have ensured that the target gene into imported and engineered bacteria successfully constructed. In the next experiment, we tested the changes of salinity tolerance ability of the engineered bacteria.

Engineering bacteria: e coli.BL21 (DE3)-pET-28a (+)-LS21sufB saline-alkali plate medium pH is 8.5.After induction, the bacterial solution is diluted to 10-2, and the inoculation amount in each cell is 30μL.

![](https://static.igem.wiki/teams/4835/wiki/engneer/result.png)

In order to verify the saline-alkali tolerance of LS21sufB engineering bacteria, four media with different sodium chloride gradients were cultured and observed. Compared with E coli.BL21 (DE3) wild-type, no-load and uninduced engineering bacteria, it was found that the engineering bacteria significantly improved the saline-alkali tolerance. The highest saline-alkali tolerance values of E coli.BL21 (DE3)-pET-28a (+)-LS21sufB were 10%NaCl and pH=8.5./p>

![](https://static.igem.wiki/teams/4835/wiki/avater/avatar.png)

![](https://static.igem.wiki/teams/4835/wiki/avater/walking.gif)

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Experiments | XJU-China
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Validation of plasmid construction by enzymatic digestion experiments
Preparation of Escherichia coli receptor cells
Transformation of plasmids
Screening of positive clones
Expression of target genes
Protein detection
E coli. Dh5α-pET-28a(+)-LS21sufB salt concentration shake flask test
Determination of salinity tolerance of LS21sufB engineering bacteria

# Validation of plasmid construction by enzymatic digestion experiments

## experimental step：

Reaction system:
total volume 20 μL
NcoⅠ:1μL
HindⅢ:1μL
10×H Buffer:2μL
Plasmid DNA: 10μL
ddH2O :6μL

## Reaction conditions:

Temperature 37°C, enzyme digestion for 3 hours.
1. Agarose gel electrophoresis to detect the results of enzyme digestion
Prepare 1% agarose gel (containing 0.5μg/mL nucleic acid dye), electrophoresis at 90V for 30 min.
2. UV transilluminator to observe the results (lane 1: original plasmid; lane 2: plasmid digested by restriction endonuclease.

# Preparation of Escherichia coli receptor cells

## Experimental Steps:

### Preparation:

Configuration and sterilization of 0.05mol/L CaCl2 solution, 0.05mol/L CaCl2 solution containing 15% glycerol; E. coli BL21, E. coli S17-1 plate delineation to ensure single colonies
1、Pick single colony from the plate and incubate it in 5ml LB medium overnight.
2, Take 100ul of bacterial solution and add it into 10mL LB medium. Shake at 220 rpm for about 3-5 hours to make OD600 between 0.4~0.5.
3、Sub-transfer the bacterial solution into 1.5ml centrifuge tubes (one tube of 10ml bacterial solution can be divided into about 6 tubes of 1.5ml centrifuge tubes), place on ice for 10 minutes, and centrifuge at 3000g for 10 minutes at 4℃.
4. Discard the supernatant, gently suspend the cells with 200 μL of pre-cooled 0.05 mol/L CaCl2 solution, and place on ice for 10 min.
The cells were suspended in 200 μL of pre-cooled 0.05 mol/L CaCl2 solution, placed on ice for 30 min, and then centrifuged at 3000g for 10 min at 4℃.
5. Discard the supernatant, add 200 μL of pre-cooled 0.05 mol/L CaCl2 solution containing 15% glycerol, gently suspend the cells, and place on ice for a few minutes to form a sensory cell suspension. The cells were kept on ice for a few minutes to form a sensory cell suspension. Plasmid transformation

# Transformation of plasmids

## Experimental steps:

1, 10μL of recombinant plasmid was mixed with 50μL of susceptible E. coli BL21
2、Mix the recombinant plasmid with the receptor cells in an ice bath for 30min.
3、Hot excitation at 42℃ for 90s, and then ice bath for 2-3min.
4、Add 500μl of LB medium, 37℃, 220rpm, incubate for 1-2h.
5、Take 200μL and spread it evenly on LB solid medium containing 100μg/ml Kana, incubate at 37℃ inverted overnight.

# Screening of positive clones

## Experimental steps:

1 Pick the overnight cultured colonies (2 parallels per plate) into the LB liquid medium containing 100μg/ml Kana, 37℃, 150rpm, overnight culture, prepare the seed solution, and then carry out bacterial liquid PCR for verification.
2 Bacterial liquid PCR: take 2μL of bacterial liquid on the ultra-clean bench, add 100μL of ddH2O, mix well, and boil in a 100℃ water bath for 15min.

3 20μL system
Bacterial liquid: 4μL
Upstream primer: 1μL
Downstream primer: 1μL
Master mix:6μL
ddH2O:8μL
(Upstream primer: ATGGCAAGCGAAGAAATGGAAC)
Downstream primer: TTAACCCACTGCGCCTTCAAG)
PCR Parameters.
95°C pre-denaturation:5min➔main cycle 94°C:30s➔55°C:30s➔72°C:2min cycle 30 times➔72°C:10min➔4°C:50min

# Expression of target genes

The activated engineering bacteria were pre-cultured 37°C overnight. Growth at 1% inoculum reached 0.6 ≤ OD600 ≤ 0.8 at 37°C. 0.1 mM IPTG was added (no IPTG was added to the control group) and growth was carried out at 30°C for 2 hrs. The experimental group with OD600 ±1.0, diluted the above bacterial solution with LB at 0.01% inoculum. The growth was observed every 12h by inoculating into 8% NaCl, pH=8 LB plates at an inoculum of 50μl.

# Protein detection

Take 30ml of the induced bacterial solution,at 4℃,,centrifuge for 5min (5000g). Discard the supernatant and add 1% lysozyme containing 1mg/ml. Then freeze and thaw three times: at 37℃ for 5min, and then at -80℃ for 5min. ultrasonic cell crusher was used to sonicate for 3s, stop for 3s, and work for 20min (power: 40%, 360W), and the obtained solution was centrifuged at room temperature for 10min, and the supernatant was taken as the upper sample solution, and electrophoresis was performed.

# Salt concentration shake flask test

Procedure: inoculate the induced bacterial solution at 1% inoculum into 250 ml conical flasks. (100 ml sample volume)

Control group: A wild-type Dh5α (WT) medium without kana, B empty (EV) medium with kana.

Experimental group: E coli. Dh5α-pET-28a(+)-LS21sufB(LSD) medium with kana.

Note: The addition of the antibiotic kana to the medium of the empty and experimental groups serves to prevent contamination by stray bacteria.

# Determination of salinity tolerance of engineering bacteria

Engineering bacteria: E coli.BL21(DE3)-pET-28a(+)-LS21sufB .

Saline plate medium pH 8.5, diluted to 10-2 after induction, 30μL per compartment.

![](https://static.igem.wiki/teams/4835/wiki/avater/avatar.png)

![](https://static.igem.wiki/teams/4835/wiki/avater/walking.gif)

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Results | XJU-China
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[Interaction with participating teams](#section1)
1. Certification
2. Invitation
3.Videos
4.Cooperation
Communication with public
Conference Communication

# OVERVIEW

Relying on such an engineering model, we succeeded in obtaining universal gene originals in non-model strains that can improve the salinity and base resistance of model strains.

The experiment proves that our engineering is very successful, and such an experimental idea can also be used for mining other elements, we welcome every teams who want to do this work to communicate and use this method.

I will describe the method in detail and show the success we achieved in this page.

![](https://static.igem.wiki/teams/4835/wiki/engneer/lc2.png)

# 1.Model Result

There were two up-regulated differentially expressed genes, sufB (iron-sulfur cluster scaffolding complex subunit) and mnmA (tRNA-specific 2-thiouracilase), and no down-regulated differentially expressed genes among the six strains; there was one up-regulated differentially expressed protein, purM among the six strains.

The six strains of salinity-tolerant bacteria were screened for differentially expressed genes on both the transcriptome and proteome without differentiating between up- and down-regulation, and found that the six strains had 19 differentially expressed genes, the six strains had 21 differentially expressed proteins, and the six strains had five differentially expressed genes on both the transcriptome and proteome.（details are shown in model section）

In summary, a total of 40 genes with high likelihood were screened, and finally, in combination with the metabolomic data, the six most probable universal antisaline elements expressed at transcriptional, translational, and metabolic levels by saline-tolerant bacteria of different genera in response to different saline environments were listed. The results are as follows.

![](https://static.igem.wiki/teams/4835/wiki/engneer/eng-liucheng.png)

Northwestern University Invitation

![](https://static.igem.wiki/teams/4835/wiki/engneer/t5-9.png)

![](https://static.igem.wiki/teams/4835/wiki/engneer/t5-10.png)

![](https://static.igem.wiki/teams/4835/wiki/engneer/dna-result.png)

The gene we found

# 2.Successful of wet lab

Through the above steps, we have found sufB, it can enhance the ability of E.coli to tolerant the saline and base environment.

Engineering bacteria: e coli.BL21 (DE3)-pET-28a (+)-LS21sufB saline-alkali plate medium pH is 8.5.After induction, the bacterial solution is diluted to 10-2, and the inoculation amount in each cell is 30μL.

![](https://static.igem.wiki/teams/4835/wiki/engneer/result.png)

In order to verify the saline-alkali tolerance of LS21sufB engineering bacteria, four media with different sodium chloride gradients were cultured and observed. Compared with E coli.BL21 (DE3) wild-type, no-load and uninduced engineering bacteria, it was found that the engineering bacteria significantly improved the saline-alkali tolerance. The highest saline-alkali tolerance values of E coli.BL21 (DE3)-pET-28a (+)-LS21sufB were 10%NaCl and pH=8.5./p>

![](https://static.igem.wiki/teams/4835/wiki/avater/avatar.png)

![](https://static.igem.wiki/teams/4835/wiki/avater/walking.gif)

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