iGEM 2025 — Team xju-china

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{% block title %}Notebook | XJU-China{% endblock %}
{% block lead %}Describe how and why you chose your iGEM project.{% endblock %}
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![snow mountain](https://static.igem.wiki/teams/5543/jiazaixueshan/jiazairenbeijing.webp)
![Loading...](https://static.igem.wiki/teams/5543/loader/loader/aaa.webp)

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[Wet Experiments](#section1)

[Dry Experiments](#section2)

[Easter egg](#section3)

[Retrospect](#section4)

Notebook

Wet Experiments

### March 31 – April 14

#### Plasmid resource mining

1. sampling
2. strain isolation and purification
3. Species and Genus Identification of Bacterial Strains
4. Plasmid extraction

![#](https://static.igem.wiki/teams/5543/notebook/1.avif)

We are artificially extracting plasmids.

### April 15 – April 29

1. Still extracting plasmids

![#](https://static.igem.wiki/teams/5543/notebook/2.avif)

Do a great job, workstation—we know you can do it!

### April 30 – May 12

1. Yet again, we're extracting plasmids.

![#](https://static.igem.wiki/teams/5543/notebook/3.avif)

We're currently centrifuging, and we'll service our workstation.

### May 12 –June 12

#### Functional testing of bacteria And Plasmid resource mining

1. During this period, we will conduct stress culture tests,including low temperature, high
   temperature, acid, alkali, and salt,on the natural plasmid-containing bacteria that were screened
   between March 31 and May 12.
2. Continue the plasmid resource mining work

![#](https://static.igem.wiki/teams/5543/notebook/4.avif)

The bacteria under "inspection" are currently in shake flasks.

### June 12 – July 12

#### Functional testing of bacteria

1. During this period, we cultured natural bacteria containing plasmids under stress conditions,
   including low temperature, high temperature, acid, alkali, and salt.

![#](https://static.igem.wiki/teams/5543/notebook/5.avif)

We are currently inoculating bacteria into the culture medium

### June 12 – August 12

#### Robustness testing of recombinant engineered bacteria

1. Natural plasmids were introduced into the E. coli BL21 strain to construct recombinant engineered
   bacteria, which were then subjected to stress test cultivation. The stress conditions included low
   temperature, high temperature, acid, alkali, and salt respectively.

### June 16 – July 16

#### ORF Prediction and Functional Verification

1. ORF Prediction and Functional Verification.Functional verification included stress cultivation
   tests and SDS-PAGE electrophoresis analysis.

![#](https://static.igem.wiki/teams/5543/notebook/6.avif)

We are currently using a PCR instrument to construct recombinant engineering bacteria.

### July 17 – August 17

1. Yet again，ORF Prediction and Functional Verification.

![#](https://static.igem.wiki/teams/5543/notebook/7.avif)

We are currently performing heat shock transformation.

### August 18 – September 18

1. It's finally over! ORF Prediction and Functional Verification.

Dry Experiments

### April 5 – April 12

#### Background research

1. This week we focused on some of the paperwork and the groundwork for the experiments ahead

### July 7 - July 14

#### Comparative analysis of bacterial species

1. Classify bacterial strains according to sampling locations
2. Classify bacterial strains according to genus-species relationships

#### Sequence alignment analysis

1. Complete sequence determination of plasmids
2. Sequence alignment and analysis of plasmids

### June 9 - August 11

1. ORF predictionDetermine the ORF sequences
2. Conduct analysis and comparison of the ORF sequences

![#](https://static.igem.wiki/teams/5543/notebook/8.avif)

We feel a sense of joy looking at the map we built.

Easter egg

![#](https://static.igem.wiki/teams/5543/notebook/c1.avif)

![#](https://static.igem.wiki/teams/5543/notebook/t1.avif)

A smooth and efficient spotting (Ｔ▽Ｔ)

![#](https://static.igem.wiki/teams/5543/notebook/t2.avif)

Found a misplaced pipette tip Σ(ﾟ∀ﾟﾉ)ﾉ

![#](https://static.igem.wiki/teams/5543/notebook/t3.avif)

I prepped a whole bunch of culture medium, but it ended up getting contaminated—guess I need to step up
my game with sealing and sterilization checks!(╥\_╥)

![#](https://static.igem.wiki/teams/5543/notebook/t4.avif)

I washed a deadly amount of bottles again today—seriously, why do there have to be so many?!（╯－＿－）╯╧╧

![#](https://static.igem.wiki/teams/5543/notebook/t5.avif)

Extracting plasmids is just so tedious—seriously, it’s draining.╮(︶﹏︶)╭

Retrospect

Every step of this experiment was steeped in the accumulation of "untiring efforts": In order to catch up
with the schedule, team members lingered around the workbench for several consecutive days. The unwashed
lab bottles piled up like a small mountain, and the prepared culture medium would be rendered useless by
accidental bacterial contamination in the blink of an eye. Yet the results always fell a notch short of
expectations, even the pen used to record data seemed to carry a touch of heaviness.

On several occasions, when faced with piles of samples and unsatisfactory graphs, everyone had the
thought of "should we pause for a while?" But no one actually stopped — some volunteered to work
overtime to optimize the experimental process, while others pored over numerous literatures to adjust
the extraction parameters. Even if each improvement was only a tiny step, they persisted in gathering
these trivial efforts bit by bit.

It was precisely this little bit of perseverance and repeated refusal to give up that, like the
footprints left by caravans step by step along the ancient Silk Road, turned the initial scattered
attempts into a coherent operational framework. Later, this refined process extended to broader
experimental directions. In the end, we not only achieved excellent results that exceeded expectations,
but also gained a profound insight: There is no overnight success on the road of scientific research.

Those seemingly trivial accumulations and the perseverance in the face of difficulties are just like the
perseverance of the Silk Road that crossed mountains and seas. They will eventually connect "dots" into
"lines" and expand "lines" into "planes", fostering true value through breakthroughs. This growth has
become the most precious asset on our scientific research journey.

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{% block title %}Engineering | XJU-China{% endblock %}
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![snow mountain](https://static.igem.wiki/teams/5543/jiazaixueshan/jiazairenbeijing.webp)
![Loading...](https://static.igem.wiki/teams/5543/loader/loader/aaa.webp)

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[Plasmid resource mining](#section1)

[Functional testing of bacteria](#section2)

[Establishment of Recombinant Engineered Bacteria and Functional Validation](#section3)

[ORF Prediction and Functional Verification](#section4)

Engineering

Plasmid resource mining

### Design

Sampling was conducted in various harsh environmental areas of Xinjiang, followed by isolation and purification of the samples.

Plasmids were extracted using the alkaline lysis method, combining the use of an automated workstation with manual extraction verification.

### Build

During sampling, corresponding layout and collection methods were employed based on sample characteristics. For soil samples, diagonal and checkerboard patterns were used with sterilized sampling bags for surface soil and sediment collection.

Water samples were collected using sterile sampling bottles to avoid contamination, with appropriate preservatives added onsite to stabilize microbial communities.

Camel milk and fecal samples were sealed in sterile centrifuge tubes or sampling bags and maintained at low temperatures to ensure rapid transfer.

The samples were diluted, and the bacterial strains in the resulting leachate were isolated using the dilution plating method. Single colonies grown were picked and subjected to monoclonal culture using the streak plate method.

16S rRNA gene sequencing was performed on the purified bacterial strains. Meanwhile, statistics were collected regarding the source, species, and quantity of the strains, followed by a comparative analysis of the strains' genera and species.

### Test

A total of 2,000 bacterial strains were obtained through sampling, including 495 strains from milk samples, 182 strains from soil samples, 1078 strains from water samples, and 245 strains from fecal samples.

The collected samples were diluted to 10-4, and isolation was performed using five types of media: 2216E, Actinomycete medium, R2A, TSB, and Silicate medium, via the dilution plating method.

Single colonies were purified and cultured on LB medium using the streak plate method. Plasmids were extracted using an automated workstation.

### Learn

After extraction, a total of 38 plasmid-containing bacteria were obtained, with a plasmid-containing rate of 1.9%. In the next step, the robustness of these natural plasmid-containing bacteria will be further determined.

Functional testing of bacteria

### Design

To verify the robustness of the natural plasmid-containing bacterial strains isolated from extreme environments in Xinjiang, our team adopted a comprehensive multi-stress preliminary screening strategy.

A preliminary tolerance assessment was conducted on the wild-type bacterial strains. By observing their growth curves, colony morphology, and survival rates, we initially determined the types of stress under which the strains exhibit strong natural resistance.

### Build

We prepared culture media with different concentrations of NaCl (5%–7%), adjusted the pH to 9.0–10.0 using NaOH, and adjusted the pH to 3.5–5.0 using HCl.

For high-temperature stress, a shaking incubator with a temperature gradient of 40℃ to 45℃ was set up; for low-temperature stress, a shaking incubator with a temperature gradient of 16℃ and 18℃ was used to compare the growth performance of the strains at different temperatures.

The activated seed liquid of the wild-type bacterial strains was cultured with shaking at 37℃ and 200 rpm until the OD 600 reached approximately 0.8.

### Test

After stress test cultivation under conditions of pH 4.2, pH 9.0, 6% NaCl, 45℃, and 18℃, we found that the bacterial strain exhibits acid resistance,alkali resistance,saline-alkali resistance,low-temperature resistance and high-temperature resistance function.

### Learn

We have identified that 8 strains containing natural plasmids exhibit robustness.

To further explore the role of plasmids in these bacteria, we constructed recombinant engineered bacteria using the extracted plasmids and conducted tests on them.

Establishment of Recombinant Engineered Bacteria and Functional Validation

### Design

Natural plasmids were introduced into E. coli BL21 engineered bacteria, and stress environment experiments were conducted with blank engineered bacteria as controls.

The stress environment was the same as that used in the natural plasmid stress test.

### Build

Before the experiment, reference sequences of the target gene family or entire genome were obtained from public databases (such as NCBI's GenBank, ENA, and UniProt). These sequences include the nucleic acid or protein sequences of the samples to be analyzed, as well as the sequences of known species used as references.

A local alignment database was constructed to compare the known plasmid sequences with plasmids of the same genus and species. Through comparative analysis of plasmid sequences, the conservation and specific differences of core functional modules (e.g., replicons, transfer elements) in their gene composition were revealed; the distribution and dynamics of functional elements such as resistance genes and mobile elements were identified. This comprehensive analysis was conducted to clarify the function, evolutionary strategy, and application value of the plasmids.

Specific primers with homologous arms were designed, and linearized recombinant fragments were obtained via PCR amplification. Subsequently, a seamless cloning kit was used, and the reaction was carried out in a PCR instrument for 50 minutes to connect the linear fragment with the linearized vector in vitro through complementary pairing of homologous arms, forming a circular recombinant vector.

The recombinant vector was introduced into E. coli BL21 competent cells via the heat shock method. After the transformed bacteria were resuscitated, they were spread on LB solid resistant plates containing kanamycin sulfate for positive clone screening. Single colonies were picked and cultured, and the correctness of the recombinant vector construction was confirmed by both colony PCR and gene sequencing.

### Test

After stress test cultivation under conditions of pH 4.5, pH 9.0, 6% NaCl, 45℃, and 18℃, we found that the recombinant engineered bacteria containing pSM4,pSM8 and other plasmids exhibited alkali resistance, acid resistance, and saline-alkali function.

### Learn

Through this round of experiments, we confirmed that plasmids play a role in enhancing the robustness of the host bacteria. Next, we conducted further verification to identify which specific fragments of the plasmids are responsible for this function.

ORF Prediction and Functional Verification

### Design

ORF (Open Reading Frame) prediction was performed on plasmids with robustness function. Recombinant engineered bacteria were constructed using ORFs with robustness function, followed by stress test cultivation and SDS-PAGE analysis.

### Build

ORF prediction was performed to convert abstract DNA sequences into a specific list of functional genes, thereby systematically revealing the composition of plasmid replication and partitioning mechanisms, adaptive genes such as antibiotic resistance genes, and mobile elements like conjugative transfer elements.

All sequences were codon-optimized according to the E. coli expression system to obtain target sequences encoding mature proteins. The sequences were submitted to Youkang Biotechnology Co., Ltd. for chemical synthesis and then cloned into the pET-28a(+) vector. The recombinant plasmids were subjected to double enzyme digestion verification using an ApaI-XhoI double enzyme digestion reaction system at 37℃ for 3 hours.

The stress cultivation test was conducted in the same manner as that for the aforementioned recombinant engineered bacteria.

The bacteria containing the recombinant plasmids were cultured in a stress environment corresponding to the plasmid function. When the OD₆₀₀ of the bacterial culture reached 0.8–1.0, the recombinant engineered bacteria were picked and inoculated into 30 mL of LB liquid medium containing kanamycin (Kana), followed by overnight culture at 37℃ and 150 rpm to prepare the seed liquid.

The seed liquid was transferred to LB liquid medium containing Kana at an inoculation amount of 1%, and cultured at 37℃ with shaking at 200–220 rpm until the OD₆₀₀ reached approximately 0.6–0.8, then induced with IPTG.

The induced bacterial culture was centrifuged, the supernatant was discarded, and PBS and 5× loading buffer were added at a ratio of 4:1, mixed well, and boiled at 100℃ for 15 minutes. The supernatant was retained for subsequent SDS-PAGE analysis.

### Test

Through this round of experiments, we confirmed that plasmids play a role in enhancing the robustness of the host bacteria.

### Learn

Through this experiment, we found that ORF3 and ORF7 derived from pSM4, as well as ORF5 derived from pSM18, all exhibit tolerance under 6% NaCl and pH=9 environments.ORF12 shows tolerance under both pH=4.2 and pH=9 environments.

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{% extends "layout.html" %}
{% block title %}Results | XJU-China{% endblock %}
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![snow mountain](https://static.igem.wiki/teams/5543/jiazaixueshan/jiazairenbeijing.webp)
![Loading...](https://static.igem.wiki/teams/5543/loader/loader/aaa.webp)

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[Plasmid resource mining](#section1)

[Functional testing of bacteria](#section2)

[Robustness testing of recombinant engineered bacteria](#section3)

[ORF Prediction and Functional Verification](#section4)

Results

Plasmid resource mining

After sampling, screening natural bacteria, and conducting species identification on them, we found that the 2,000 bacterial strains collected belong to 120 genera and a total of 286 species.

![Species and genus distribution results of collected bacterial strains](https://static.igem.wiki/teams/5543/results/result1.webp)

The extracted plasmids were verified by agarose nucleic acid electrophoresis, and the results are shown in the figure.

![Agarose nucleic acid electrophoresis results of extracted plasmids](https://static.igem.wiki/teams/5543/results/result2.webp)

| Lane Number | Plasmid Number | Lane Number | Plasmid Number | Lane Number | Plasmid Number |
| --- | --- | --- | --- | --- | --- |
| 1 | pRM 1 | 14 | pRM 14 | 27 | pEM 2 |
| 2 | pRM 2 | 15 | pRM 15 | 28 | pEM 3 |
| 3 | pRM 3 | 16 | pRM 16 | 29 | pWM 1 |
| 4 | pRM 4 | 17 | pRM 17 | 30 | pWM 2 |
| 5 | pRM 5 | 18 | pRM 18 | 31 | pKM1 |
| 6 | pRM 6 | 19 | pRM 19 | 32 | pKM2 |
| 7 | pRM 7 | 20 | pSM 1 | 33 | pKM3 |
| 8 | pRM 8 | 21 | pSM 2 | 34 | pKM6 |
| 9 | pRM 9 | 22 | pSM 3 | 35 | pCM1 |
| 10 | pRM 10 | 23 | pSM 4 | 36 | pCM2 |
| 11 | pRM 11 | 24 | pSM 5 | 37 | pCM3 |
| 12 | pRM 12 | 25 | pSM 6 | 38 | pCM4 |
| 13 | pRM 13 | 26 | pEM 1 |  |  |

Functional testing of bacteria

Due to time constraints, we only conducted experimental verification on 30 out of the 38 screened natural plasmid-containing bacterial strains.

Salt stress test: The test was carried out under the culture condition with a NaCl concentration of 7%. The growth curves of 30 plasmid-containing bacterial strains in LB medium with 7% NaCl are shown in Figure 1. In the environment containing 7% NaCl, all 30 host bacterial strains grew well, and strain No. 2-8-4 had the highest growth amount.

![Growth curves of bacterial strains under 7% NaCl concentration (Part 1)](https://static.igem.wiki/teams/5543/results/1-1.webp)
![Growth curves of bacterial strains under 7% NaCl concentration (Part 2)](https://static.igem.wiki/teams/5543/results/1-2.webp)

Alkali stress test: The growth ability in an alkaline environment with pH=10 was tested. In the medium with pH=10, all 30 plasmid-containing bacterial strains could grow normally, and there were differences in the growth amount measured by OD600.

The test results under the alkali stress of pH 10 are shown in Figure 2, and strain No. 2-8-4 had the highest growth amount.

![Growth curves of bacterial strains under pH=10 alkaline stress (Part 1)](https://static.igem.wiki/teams/5543/results/2-1.webp)
![Growth curves of bacterial strains under pH=10 alkaline stress (Part 2)](https://static.igem.wiki/teams/5543/results/2-2.webp)

Acid stress test: The test was conducted in an acidic environment with pH=4.9. The growth amounts of the 30 plasmid-containing bacterial strains are shown in Figure 3. Strain No. Y4 had the highest growth amount.

![Growth curves of bacterial strains under pH=4.9 acidic stress (Part 1)](https://static.igem.wiki/teams/5543/results/3-1.webp)
![Growth curves of bacterial strains under pH=4.9 acidic stress (Part 2)](https://static.igem.wiki/teams/5543/results/3-2.webp)

Low-temperature stress test: The test was performed under the culture conditions of 16℃ and 18℃. Under the culture condition of 16℃, 14 bacterial strains including YH4, 2-3-44 and M6-66 grew normally, and their growth curves are shown in Figure 4.

In the low-temperature environment of 16℃, strain Y12 had the highest growth amount. Since 16 plasmid-containing bacterial strains could not grow at 16℃, the test was conducted at 18℃, and the test results at 18℃ are shown in Figure 5.

![Growth curves of bacterial strains under 16℃ low-temperature stress](https://static.igem.wiki/teams/5543/results/4-1.webp)
![Growth curves of bacterial strains under 18℃ low-temperature stress](https://static.igem.wiki/teams/5543/results/5.webp)

High-temperature stress test: Initially, a high temperature of 45℃ was set to test the high-temperature resistance of the plasmid-containing bacterial strains. In the test environment of 45℃, the 22 plasmid-containing bacterial strains are as shown in Figure 6, and strain No. 2-8-4 had the highest growth amount.

![Growth curves of bacterial strains under 45℃ high-temperature stress (Part 1)](https://static.igem.wiki/teams/5543/results/6.webp)
![Growth curves of bacterial strains under 45℃ high-temperature stress (Part 2)](https://static.igem.wiki/teams/5543/results/6-2.webp)

Robustness testing of recombinant engineered bacteria

### Verification of Transformation Results of Recombinant Engineering Bacteria

Ten natural plasmid vectors were linearized and then subjected to homologous recombination with the target gene fragment of the Kana⁺ resistance gene.

Among them, 8 natural plasmid vectors and the target gene fragment all amplified a single bright band, while 2 natural plasmids did not amplify any bands.

This indicates that the PCR system is effective, the primers specifically bind to the template, and the products can be used for subsequent homologous recombination.

After the 8 recombinant plasmids were transformed into *Escherichia coli* BL21 by heat shock, positive clones were all formed on LB plates containing Kana⁺.

The results show that homologous recombination successfully inserted the Ori (Origin of Replication) and Kana⁺ resistance gene into the natural plasmids, and the recombinant plasmids can replicate autonomously and express resistance in the host bacteria.

![Verification results of recombinant engineering bacteria transformation](https://static.igem.wiki/teams/5543/results/result3.webp)

### Results of Stress Tests

The results of the salt tolerance test are shown in Figure 7.

The test results indicate that under the salt condition of 6% NaCl concentration, after plasmid introduction, compared with the control group E. coli BL21, the growth of 7 strains increased except for the engineering bacteria containing the pRM13+K plasmid. The engineering bacteria containing the recombinant plasmid pWM2+K had the highest growth.

It is evident that plasmid pWM2 has a strong alkali tolerance function.

![Salt tolerance test results of recombinant engineering bacteria](https://static.igem.wiki/teams/5543/results/7.webp)

The results of the alkali tolerance test are shown in Figure 8.

The test results indicate that under the alkaline condition of pH=9, after plasmid introduction, compared with the control group E. coli BL21, the growth of all 8 strains increased. Among them, the growth of engineering bacteria containing recombinant plasmids pWM1+K, pRM4+K, and pRM7+K increased most significantly.

The growth of other engineering bacteria containing recombinant plasmids was relatively similar to that of the control group E. coli BL21. It is推测 that plasmids pWM1, pRM4, and pRM7 have strong alkali tolerance functions.

![Alkali tolerance test results of recombinant engineering bacteria](https://static.igem.wiki/teams/5543/results/8.webp)

The results of the acid tolerance test are shown in Figure 9.

The test results indicate that under the alkaline condition of pH=4.15, after plasmid introduction, compared with the control group E. coli BL21, the growth of 7 strains increased except for the engineering bacteria containing the pRM13+K plasmid. Among them, the growth of engineering bacteria containing recombinant plasmids pRM4+K and pRM6+K increased most significantly.

It is evident that plasmids pRM4 and pRM6 have strong acid tolerance functions.

![Acid tolerance test results of recombinant engineering bacteria](https://static.igem.wiki/teams/5543/results/9.webp)

The results of the low-temperature tolerance test are shown in Figure 10.

The test results indicate that under the low-temperature condition of 18℃, after plasmid introduction, compared with the control group E. coli BL21, the growth of all 8 engineering strains was lower than that of the control.

It is speculated that none of the 8 plasmids used in this study have low-temperature tolerance ability.

![Low-temperature tolerance test results of recombinant engineering bacteria](https://static.igem.wiki/teams/5543/results/10.webp)

The results of the high-temperature tolerance test are shown in Figure 11.

The test results indicate that under the high-temperature condition of 45℃, after plasmid introduction, compared with the control group (E. coli BL21), the growth of 7 out of the engineering bacteria containing recombinant plasmids increased, except for those harboring the pWM1+K and pRM+K plasmids.

Among them, the engineering bacterium containing the recombinant plasmid pRM4+K showed the most significant increase in growth.

It is speculated that plasmid pRM4 has a strong high-temperature tolerance function.

![High-temperature tolerance test results of recombinant engineering bacteria](https://static.igem.wiki/teams/5543/results/11.webp)

ORF Prediction and Functional Verification

### ORF Prediction Results

The functions of proteins encoded by plasmid sequences that appear multiple times in different plasmids mainly fall into four categories:

The first category is involved in the replication and repair of plasmid DNA, such as sequences encoding replication initiation proteins, plasmid recombination enzymes, DNA repair and recombination proteins, and tRNA ligases.

The second category includes sequences that participate in encoding the host's physiological metabolic activities and energy generation and metabolism. These sequences have functions of encoding triosephosphate isomerases, regulator aspartate phosphatase E, alpha-1,3-glucan synthases, and phosphoglycolate phosphatases.

The third category of encoded proteins mainly functions in substance transport and signal transduction. Examples include sequences encoding maltose/maltodextrin transport system permease protein MalF, proteins of the NRT1/PTR family, and phosphate import ATP-binding proteins.

The fourth category consists of ORFs (Open Reading Frames) that encode proteins with specific functions, such as probable F-box proteins and small heat shock protein C4. F-box proteins are important components involved in the substrate ubiquitination process and participate in the assembly of ubiquitin ligases. In contrast, small heat shock protein C4 is induced to express when cells are exposed to various stresses (such as high temperature, oxidative stress, etc.) and plays a role in protecting cells from stress damage.

From the basic physiological metabolism of bacteria to stress responses, ORFs with different functions are predicted to be involved in these processes. The following are some predicted results of plasmid ORFs.

| Plasmid | Strain | ORF | Coding Protein | No Significant Similarity |
| --- | --- | --- | --- | --- |
| pRM 4 | 2-8-4 | ORF9 | Putative replication protein | 7 |
| ORF8 | Kanamycin nucleotidyltransferase |
| ORF11 | Flagellar brake protein YcgR |
| ORF5 | Probable F-box protein |
| ORF6 | DNA replication and repair protein |
| pRM 6 | FH 2-3-8 | ORF9 | Protein NRT1/ PTR FAMILY | 11 |
| ORF12 | RNA polymerase II degradation factor 1 |
| ORF10 | Plasmid recombination enzyme |
| ORF1 | Maltose/maltodextrin transport system permease protein MalF |
| pRM 7 | M6-66 | ORF6 | DNA primase TraC | 3 |
| ORF1 | Plasmid recombination enzyme type 3 |
| ORF5 | Major capsid protein |
| pRM 8 | M6-69 | ORF3 | Methyl-accepting chemotaxis protein III | 3 |
| ORF4 | Mobilization protein MobL |
| ORF1 | Type II methyltransferase M.AgeI |
| ORF2 | Chromosome partitioning protein ParA |
| ORF8 | Tyrosine recombinase XerD (+ 6310-6876) |
| ORF9 | Transposase InsF for insertion sequence IS3A (+ 346-693) |
| pRM 13 | 2-3-44 | ORF9 | Response regulator aspartate phosphatase E | 5 |
| ORF4 | Nipped-B-like protein B |
| ORF1 | Netrin receptor DCC |
| ORF3 | Replication initiation protein |
| pWM 1 | 3-6-1 | ORF4 | Replication initiation protein | 17 |
| ORF11 | DNA replication and repair protein |
| ORF5 | Putative O-methyltransferase MUL\_4520 |
| ORF16 | Endoglucanase 16 |
| ORF9 | Triosephosphate isomerase |
| pWM 2 | YH4 | ORF17 | Triosephosphate isomerase | 19 |
| ORF12 | Restriction of telomere capping protein 5 |
| ORF19 | Transcription antitermination protein NusB |
| ORF13 | Putative O-methyltransferase MUL\_4520 |
| ORF3 | Endoglucanase 16 |
| pSM 4 | YM-5 | ORF11 | Replication initiation protein | 18 |
| ORF7 | Transposon Tn552 |
| ORF3 | Plasmid-partitioning protein ParA |
| ORF20 | Putative toxin HigB3 |
| ORF23 | Antitoxin HigA1 |

### SDS-PAGE analysis and double enzyme digestion verification Result

Based on the frequency of ORF occurrence and their functions, we selected 12 ORFs.

After SDS-PAGE analysis and Restriction Enzyme Verification, we constructed recombinant engineering bacteria.

We conducted stress test cultivation under three conditions: pH=4.15, pH=9, and 6% NaCl.

The protein gel images are shown in Figures a, b, and c.

![SDS-PAGE analysis results (Figure a)](https://static.igem.wiki/teams/5543/results/figure-a.webp)
![SDS-PAGE analysis results (Figure b)](https://static.igem.wiki/teams/5543/results/figure-b.webp)
![SDS-PAGE analysis results (Figure c)](https://static.igem.wiki/teams/5543/results/figure-c.webp)

| Figure | Lane | Protein |
| --- | --- | --- |
| Figure a | Lane 1 | Presumptive replication protein, uninduced |
| Lane 2 | Presumptive replication protein, induced |
| Lane 3 | Chromosome partitioning protein, uninduced |
| Lane 4 | Chromosome partitioning protein, induced |
| Lane 5 | Main partitioning protein parA, uninduced |
| Lane 6 | Main partitioning protein parA, induced |
| Lane 7 | Induced head protein, uninduced |
| Lane 8 | Induced head protein, induced |
| Figure b | Lane 1 | Small heat shock protein, uninduced |
| Lane 2 | Small heat shock protein, induced |
| Lane 3 | Replication initiation protein, uninduced |
| Lane 4 | Replication initiation protein, induced |
| Lane 5 | Induced Rip (plasmid replication initiation protein), uninduced |
| Lane 6 | Induced Rip (plasmid replication initiation protein), induced |
| Lane 7 | Induced Rip (plasmid replication initiation protein), uninduced |
| Lane 8 | Induced Rip (plasmid replication initiation protein), induced |
| Figure c | Lane 1 | DNA replication and repair protein, uninduced |
| Lane 2 | DNA replication and repair protein, induced |
| Lane 3 | Triose phosphate isomerase, uninduced |
| Lane 4 | Triose phosphate isomerase, induced |
| Lane 5 | Small heat shock protein, uninduced |
| Lane 6 | Endoglucanase, induced |
| Lane 7 | Endoglucanase 16, uninduced |
| Lane 8 | Endoglucanase 16, induced |

The results of double enzyme digestion verification are shown in the figure.

![Double enzyme digestion verification results of recombinant plasmids](https://static.igem.wiki/teams/5543/results/result4.avif)

### Results of Stress Tests

After stress tests cultivation under pH=4.15, pH=9, and 6% NaCl conditions, we have obtained the following new findings:

1. ORF3 and ORF7 derived from pSM4 both exhibit tolerance under 6% NaCl, pH=4.15 and pH=9 environments, as shown in Figure 12 and Figure 13 respectively.
2. ORF5 derived from pRM18 exhibits tolerance under 6% NaCl and pH=9 environments, as shown in Figure 13 and Figure 14 respectively.
3. ORF10 derived from pRM17 exhibits tolerance under pH=4.15 and pH=9 environments, as shown in Figure 13 and Figure 14 respectively.

![Stress test results for ORF3](https://static.igem.wiki/teams/5543/results/12-1.webp)
![Stress test results for ORF7](https://static.igem.wiki/teams/5543/results/12-2.webp)
![Comparison of stress tolerance for ORF3 and ORF7](https://static.igem.wiki/teams/5543/results/genggai.avif)

![Stress test results for ORF5 under 6% NaCl](https://static.igem.wiki/teams/5543/results/13-1.webp)
![Stress test results for ORF5 under pH=9](https://static.igem.wiki/teams/5543/results/13-2.webp)
![Stress test results for ORF5 under pH=4.15](https://static.igem.wiki/teams/5543/results/13-3.webp)

![Stress test results for ORF10 under pH=4.15](https://static.igem.wiki/teams/5543/results/14-1.webp)
![Stress test results for ORF10 under pH=9](https://static.igem.wiki/teams/5543/results/14-2.webp)

![Comparison of stress tolerance for various ORFs under pH=9](https://static.igem.wiki/teams/5543/results/15-1.webp)
![Comparison of stress tolerance for various ORFs under pH=4.15](https://static.igem.wiki/teams/5543/results/15-2.webp)

In contrast, ORF2, ORF4, ORF6, ORF8, ORF9, ORF11 and ORF12 all exhibit good tolerance under the alkaline condition of pH=9. ORF1 exhibits good tolerance under the acidic condition of pH=4.15.

![Stress test results for ORFs under pH=9 (Part 1)](https://static.igem.wiki/teams/5543/results/16-1.webp)
![Stress test results for ORFs under pH=9 (Part 2)](https://static.igem.wiki/teams/5543/results/16-2.webp)
![Stress test results for ORF1 under pH=4.15](https://static.igem.wiki/teams/5543/results/16-3.webp)

{% endblock %}