iGEM 2025 — Team shsid

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"title": "",
"content": '

| **PROJECT ACTIVITY** | **SPECIFIC ACTIVITY** | **START DATE** | **END DATE** | **TOTAL TIME**  **(WEEKS)** |
| --- | --- | --- | --- | --- |
| **Recruiting Team Members through Lectures and Tryouts** | N/A | 2024/12/01 | 2025/01/18 | 7 |
| **Development of Project Plan** | N/A | 2025/02/01 | 2025/02/27 | 3 |
| **Wet Lab Activities** | Safety Training | 2025/08/18 | 2025/08/18 | 4 |
| Plasmid Construction, PCR, and Extraction, and Transfer (to *E. coli*) | 2025/08/19 | 2025/08/21 |
| *E. coli* BL21 & Protein Experiments | 2025/08/20 | 2025/08/23 |
| Chromium Resistance Experiments | 2025/08/21 | 2025/08/24 |
| Chromium Detection Experiments | 2025/08/24 | 2025/08/25 |
| Quantification of Results | 2025/08/24 | 2025/08/25 |
| Figure-Making and Additional Research | 2025/08/25 | 2025/08/31 |
| Wiki Writing | 2025/08/26 | 2025/09/15 |
| **Dry Lab Activities** | Promotion Video Planning/Filming | 2025/08/05 | 2025/08/11 | 4 |
| Project Introduction, Background Research, Surveying for Initial Data | 2025/08/18 | 2025/08/19 |
| Prepare Interview Speeches | 2025/08/19 | 2025/08/22 |
| Business Plan Writing | 2025/08/20 | 2025/08/22 |
| Interviewing | 2025/08/21 | 2025/08/22 |
| Education Preparation and Activities | 2025/08/23 | 2025/08/25 |
| Official Account Writing | 2025/08/24 | 2025/08/25 |
| BP Revising | 2025/08/25 | 2025/09/10 |
| **Hardware Development** | Introduction Meeting | 2025/08/18 | 2025/08/18 | 4 |
| Creation of Blueprint and Purchasing Parts | 2025/08/19 | 2025/08/25 |
| Refining Model | 2025/08/22 | 2025/08/25 |
| Creating Sensory System and Physical Model | 2025/08/26 | 2025/08/26 |
| Testing of Final Product | 2025/09/07 | 2025/09/15 |
| **Art & Design Activities** | Crouton Logo Creation + Official Art | 2025/08/01 | 2025/08/05 | 3 |
| Team Uniform Design | 2025/08/18 | 2025/08/20 |
| Official Account Post Designs | 2025/08/24 | 2025/08/26 |
| Crouton Project PPT Design | 2025/09/10 | 2025/09/20 |
| **Team Activities** | Project PPT Making | 2025/08/26 | 2025/09/20 | 3 |
| Team Photos | 2025/08/26 | 2025/08/26 |

* **Wet Lab Notes**

1. **Plasmid Construction, PCR, and Extraction, and Transfer (to *E. coli*)**
2. **Protein Purification Protocol**
3. **Chromium Resistance Experiments**
4. **Chromium Detection Experiments**
5. **Quantification of Results**
6. **Hardware Development**

'}]%}
{% block title %}Notebook{% endblock %}
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![](https://static.igem.wiki/teams/5737/banner/v1/project.webp)

PROJECT

NOTEBOOK

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{% set table\_data = [{
"title": "Overview",
"content": '

**Heavily-contaminated effluent from industry
leftovers threatens both human and environmental health.** Among the toxic carcinogens is hexavalent chromium
[Cr (VI)], a highly toxic metallic element. It is highly soluble in water across a wide range of pH conditions,
granting it the mobility to easily contaminate water sources and soil. Hexavalent chromium may be reduced to a less
toxic trivalent chromium [Cr (III)], where it isn’t as toxic or soluble. Accordingly, the two crucial steps to reduce
chromium contamination are chromium detection and chromium degradation. As a result, one detection plasmid
(pET28a-Chr-T7-amilCP) and three degradation plasmids (pSC101-ChrR, pSC101-YieF, and pSC101-ChrR-YieF) were designed,
built, tested, and then reflected.

'
},{
"title": "Part 1: pET28a-chr-T7-amilCP",
"content": '',
"children": [{
"title": "Design 1",
"content": '

pET28a-chr-T7-amilCP is composed of four parts: the
plasmid backbone pET28a, chromium-biosensor regulatory module chr, a strong promoter and signal amplifier T7, and a
blue pigment protein amilCP.

**pET28a** is the widely used plasmid in
the pET (Plasmid for *E. coli* T7 expression) series, containing multiple features that aid the expression of
recombinant proteins in *E. coli*. The pET28a vector contains the selective marker kan, allowing the vector and
thus the bacteria to be resistant to the antibiotic kanamycin, providing selective pressure, and allowing only
bacteria with said selective marker to survive on kanamycin plates. Furthermore, pET28a encodes 6x histidine tags on
the gene, which can bind strongly to either Nickel (Ni2+) or Cobalt (Co2+). Additionally, the
gene contains multiple cloning sites with numerous restriction sites, allowing flexible insertion of genes of
interest. The lac operator sequence sits close to the T7 promoter, preventing leaky expression from occurring, thus
indicating the strong efficacy of pET28a as a vector.

**Chr** is a regulatory module that
controls the transcription of downstream genes.

![descript](https://static.igem.wiki/teams/5737/engineer/1.webp)

**Figure 1. chrB Gene Without Cr(VI) Present, Focusing On: p\_chr Operator, chrB Gene, and T7 Promoter in the
Regulatory Module chr**

Chr is composed of a p\_chr promoter+operator, which
recruits T7-RNA-polymerase that is vital for transcription to begin when activated, and a consistently expressed
repressor-producing gene, chrB, that codes for the repressor ChrB to inactivate the p\_chr operator when Cr(VI) is not
present.

![descript](https://static.igem.wiki/teams/5737/engineer/2.webp)

**Figure 2. chrB Gene with Cr(VI) Present, Focusing On: p\_chr Operator, chrB Gene, and T7 Promoter in the
Regulatory Module chr**

Repressor ChrB protein holds high affinity for the
promoter+operator sequence p\_chr, and binds to it when Cr(VI) is not prevalent. This binding inactivates p\_chr and its
ability to recruit **T7-RNA polymerase**(**T7-RNAP**). As only T7-RNAP could bind to the T7
promoter, the lack of T7-RNAP suppresses transcription of downstream genes. The allosteric property of ChrB repressor
protein is activated when it binds to Cr(VI) ions(CrO42-,
Cr2O74-), which induces a conformational change that causes Cr(VI)-bound ChrB\'s
dissociation from the operator. This derepression of the operator allows the initiation of transcription for
downstream genes when T7-RNAP is recruited and binds to the T7 promoter.

The **T7 promoter** is a strong promoter
sequence that is **only** recognized by T7-RNAP and is not recognized by the bacterial host\'s own RNAP.
As an extremely strong amplifier, the T7 promoter can cause target genes to be transcribed so efficiently that they
comprise up to 30% of the total cellular mRNA within a short period. Consequently, the expressed protein can account
for more than 50% of the total cellular protein after only a few hours of induction.

The **amilCP gene** came from***Acropora millepora,*** and encodes a chromoprotein called amilCP. When the gene is expressed, the
resulting protein is naturally blue-purple without needing external aid for activation, producing a direct and visible
output.

![D:/LYJ/2025-iGEM/李琦-构建可还原六价铬毒性的大肠杆菌工程菌株/最终实验结果汇总/质粒图谱/pET28a-chr-T7-amilCp Map.pngpET28a-chr-T7-amilCp Map](https://static.igem.wiki/teams/5737/engineer/3.webp)

**Figure 3. Plasmid Map of pET28a-chr-T7-amilCp**

'
},{
"title": "Build 1",
"content": '

1. **pET28a Vector Extraction &
   Linearization**

The plasmid **pET28a** was extracted
from bacterial culture, underwent Double Enzyme Digestion by adding XhoI and BamHI restriction endonucleases that
recognized their respective restriction sites, and now remains as linearized plasmid DNA. We then used an
Ultramicro-volume spectrophotometer (ZuoFei) to test the plasmid DNA\'s concentration (in ng/μL) and purity (in the
A260/A280 ratio, or the sample\'s UV light absorbance rate at 260nm divided by that value at 280nm).

![](https://static.igem.wiki/teams/5737/engineer/table-1.webp)

**Table 1. Concentration and Purity of pET28a
Plasmid**

Through repeated testing of 7 batches of plasmid DNA,
we chose two batches with relatively high concentration and purity, being batches 1 and 3. The concentration of these
two batches was respectively nearly double and triple that of the other batches, indicating their significantly higher
concentration, which will be more ideal for the homologous recombination of our desired genes. Since DNA absorbs UV
light maximally at 260nm, while proteins absorb UV light maximally at 280nm, pure DNA tends to have a ratio value of
around 1.8, while pure protein tends to have a ratio of 0.6. Thus, a far lower value may indicate protein
contamination, and a far higher value may indicate RNA contamination, or other impurities in general. While the
batches that we chose did not have the most ideal purity, we prioritized the higher concentration because the plasmid
is still required for downstream application, especially in homologous recombination, which requires enough template
DNA to ensure multiple efficient reactions. The A260/A280 values were not substantially far from 1.8, and were still
relatively closer to 1.8 (0.1~0.2 difference) compared to 0.6 (1.0~1.1 difference).

After finishing the plasmid mixture, we dyed the
plasmid using Loading Buffer, along with Big Markers (500-15000bp), and placed it in a gel electrophoresis machine.

![D:/LYJ/2025-iGEM/李琦-构建可还原六价铬毒性的大肠杆菌工程菌株/最终实验结果汇总/PCR扩增、验证、测序、菌落图/pET28a Linear 双酶切图.jpgpET28a Linear 双酶切图](https://static.igem.wiki/teams/5737/engineer/4.webp)

**Figure 4. Gel Electrophoresis Diagram for Linearized pET28a**

Figure 4 indicates a gel electrophoresis diagram of
the linearized pET28a, which is roughly around 5369 bp, as the glowing band is around the 6000 bp marker. From the
diagram above, the intensity and brightness of the pET28a band are relatively low, even in comparison with the marker.
However, its concentration remains sufficient to ensure the normal progression of subsequent experiments.

2. **Chr-T7-amilCP PCR Amplification**

Our desired gene in this experiment was
Chr-T7-amilCP, which initially experienced Polymerase Chain Reaction (PCR) amplification to produce many copies of
that gene. After PCR, we dyed the desired genes with Loading Buffer, and, along with Small (100-2000bp) and Big
(500-15000bp) Markers, we inserted the fluids into a Gel Electrophoresis Machine.

![D:/LYJ/2025-iGEM/李琦-构建可还原六价铬毒性的大肠杆菌工程菌株/最终实验结果汇总/PCR扩增、验证、测序、菌落图/Chr-T7-amilCP扩增图.jpgChr-T7-amilCP扩增图](https://static.igem.wiki/teams/5737/engineer/5.webp)

**Figure 5. Gel Electrophoresis Diagram for Gene Chr-T7-amilCP**

In Figure 5, the glowing bands indicate that the
*Chr-T7-amilCP* DNA has been successfully amplified, as its band is visualized around 1846 bp, indicating that
it follows the expected size of the gene. Thus, these successfully amplified genes will be used in homologous
recombination.

3. **Gene Fragment Recovery**

After gel electrophoresis of both the plasmid and the
gene was complete, gene fragment recovery was conducted in order to reuse the genes and plasmids in the gel for
further downstream applications. The results indicate both concentration (in ng/μL) and purity (A260/A280 ratio).

![](https://static.igem.wiki/teams/5737/engineer/table-2.webp)

**Table 2. Concentration and Purity of pET28a
Plasmid and Chr-T7-amilCP Gene After PCR and Gene Fragment Recovery**

4. **Homologous Recombination and Heat Shock Transfer**

We then set up a reaction mixture for inserting our
desired gene into the linear pET28a vector. Based on previous research, we realized that adding a small amount of
*E. coli* competent cells, such as DH5α cells, along with the plasmid and desired gene can serve as an in vivo
homologous recombination method. Here, we chose DH5α cells specifically due to their features, including the recA1
mutation, which minimizes the risk of plasmid rearrangements or deletions during propagation, allowing the inserted
fragments to have high stability. Furthermore, it also possesses the endA1 mutation, which prevents the degradation of
plasmid DNA, thereby improving the yield and quality of DNA. Additionally, it contains another genetic modification
that allows for blue/white screening, meaning easier identification of recombinant clones.

We chose to add kanamycin to the LB mixture because
we were worried that the other bacteria or unwanted colonies would contaminate the rest of the bacteria. After roughly
15 hours, the resulting cell culture was displayed as such:

![descript](https://static.igem.wiki/teams/5737/engineer/6.webp)

**Figure 6. Cell Culture Display of DH5α Bacteria with pET28a-Chr-T7-amilCP Gene**

The remaining bacteria growing on this cell culture
should contain the plasmid and genomic sequences that we desire. However, further verification is still needed, as
there is a chance that other bacteria could have spontaneously mutated to develop resistance to kanamycin, though the
probability is low. Thus, we continued with more verification testing below.

5. **Colony PCR Identification**

Five colonies from the agar plate were chosen, and we
used our pipette to dab a little bit of the colony and place it into a PCR mix, along with primers (F, forward; and R,
reverse) that define the amplification region. This mixture is then placed into a PCR machine, and the final products
are loaded into an agarose gel for gel electrophoresis. The previous gel electrophoresis used to confirm successful
Chr-T7-amilCP amplification verifies that the gene should be around 1846 bp. The glowing band results indicated below
show roughly 1846 bp as well, indicating successful insertion of the gene into pET28a, and the successful formation of
pET28a-Chr-T7-amilCP.

![D:/LYJ/2025-iGEM/李琦-构建可还原六价铬毒性的大肠杆菌工程菌株/最终实验结果汇总/PCR扩增、验证、测序、菌落图/pET28a-T7-amilCP in DH5a验证图.pngpET28a-T7-amilCP in DH5a验证图](https://static.igem.wiki/teams/5737/engineer/7.webp)

**Figure 7. Gel Electrophoresis Diagram from the Colony PCR of Bacteria in an Agar Plate**

6. **Gene Sequencing**

The extracted, recombined plasmid, along with the F
and R primers, was then sent to a genomic sequence testing company, Tsingke. The results we received are displayed as
such:

![D:/LYJ/2025-iGEM/李琦-构建可还原六价铬毒性的大肠杆菌工程菌株/最终实验结果汇总/PCR扩增、验证、测序、菌落图/pET28a-chr-T7-amilCp测序.pngpET28a-chr-T7-amilCp测序](https://static.igem.wiki/teams/5737/engineer/8.webp)

**Figure 8. Gene Sequencing of pET28a-Chr-T7-amilCP**

After gene sequencing, the desired gene has once
again been verified to be around 1846 bp, matching the results from the colony PCR gel electrophoresis as well as the
initial gel electrophoresis that confirms gene PCR amplification. Thus, through these two instances of verification,
we successfully proved that our recombined plasmid contains our desired gene, and that said recombined plasmid exists
in multiple *E. coli* cell colonies.

'
},{
"title": "Test 1",
"content":'

1. **Heat Shock Transformation of pET28a-chr-T7-amilCP into *E. coli* BL21(DE3)**

BL21(DE3) is a form of B strain *E. coli* with
a mutation on the lon and ompT genes. Removing the presence of these two proteins allows for increased protein
expression. We successfully transformed the pET28a-chr-T7-amilCP into *E. coli* BL21(DE3) using the heat shock
transformation method. After roughly 15 hours, the resulting cell culture was displayed as such:

![descript](https://static.igem.wiki/teams/5737/engineer/9.webp)

**Figure 9. Cell Culture Display of BL21(DE3) Bacteria with pET28a-Chr-T7-amilCP Gene**

After inoculation and incubation of the bacteria, we
did another Colony PCR test to check if the plasmid was successfully transferred into the competent cell. The results
show that it is around 1846bp, matching the prior PCR bp length, confirming that we successfully transferred
pET28a-chr-T7-amilCP into BL21(DE3).

![descript](https://static.igem.wiki/teams/5737/engineer/10.webp)

**Figure 10. Gel Electrophoresis Diagram from the Colony PCR of Bacteria in an Agar Plate**

2. **Cr(VI) Detection Biosensor
Analysis**

We test the effectiveness of pET28a-chr-T7-amilCP in
detecting Chromium VI at different concentrations ( 0, 10, 50, 80, 100 mg/L ). The results show that the blue color
indicates that amilCP protein is present, with the higher intensity of blue(amilCP) prevalent, which indicates the
higher chromium concentration in the solution. However, if the levels of Cr(VI) concentration in the solution are too
high, the bacteria may die and cannot express the *amilCP* gene, leading to the absence of amilCP protein,
which leads to the absence of blue color. 30mg/L is discarded due to some practical errors, which lead to some
unreliable results.

![D:/LYJ/2025-iGEM/李琦-构建可还原六价铬毒性的大肠杆菌工程菌株/最终实验结果汇总/铬检测实验/0、10、50浓度铬颜色变化.png0、10、50浓度铬颜色变化](https://static.igem.wiki/teams/5737/engineer/11.webp)

**Figure 11. pET28a-chr-T7-amilCP Turning Blue with Cr(VI) Presence**

At the same time, we checked the tubes to see if they
contained the color blue because of amilCP\'s prevalence. We transferred the different concentrations of
Cr(VI)-pET28a-chr-T7-amilCP solution onto disks and observed their color more closely.

![D:/LYJ/2025-iGEM/李琦-构建可还原六价铬毒性的大肠杆菌工程菌株/最终实验结果汇总/铬检测实验/0、10、50浓度铬颜色变化菌落图.png0、10、50浓度铬颜色变化菌落图](https://static.igem.wiki/teams/5737/engineer/12.webp)

**Figure 12. Cell Culture Display of Bacterial Strains that Expressed amilCP Protein**

The results of the petri-dishes match our results of
the tubes: the higher the chromium concentration, the higher the intensity of blue.

'
},{
"title": "Learn 1",
"content":'

In this experiment, we tested Cr(VI) detection and
proved that the detection bacteria work (changes shades of blue) when encountering different concentrations of
chromium.

Due to PCR, the concentration of the desired gene
should remain quite large, as displayed in this table above, where the concentration was over 100 ng/μL. In this case,
it is ideal if the concentration is as high as possible, as that means that many copies of the gene can be used during
homologous recombination, where this gene will have to be inserted into the plasmid pET28a. Similarly, the closer the
purity value is to 1.8, the more pure and thus the more ideal the gene\'s purity. On the other hand, the pET28a has
significantly lower concentration and slightly lower purity compared to both our desired gene as well as the
concentration and purity tests performed previously, right after plasmid extraction. This far lower concentration is
largely due to the plasmid\'s large size, causing the DNA fragments of the plasmid to diffuse more into the gel and
bind less efficiently to silica columns, and hence elute less completely, which also contributes to a lower plasmid
purity. The gel electrophoresis similarly carries agarose fragments and other substances that could contaminate the
plasmids and thus cause a lower purity.

'
}]
}, {
"title": "Part 2: pSC101-ChrR",
"content": '',
"children": [{
"title": "Design 2",
"content": '

pSC101-ChrR is composed of the
Chromate-reducing-protein producing gene ChrR combined with the plasmid backbone pSC101. ***pSC101***is *a* specialized and low-copy-numbered cloning vector that has kanamycin resistance, lacks
the *oriT* transfer origin, which preventsthe spreading of genes to other bacteria, has a specific
rhamnose-induced pathway, and has a specialized optimal replicating range at 37 °C. ***ChrR*** is
a gene that produces **chromate-reducing flavoprotein** ChrR. It is derived from *Pseudomonas
putida.* Its key characteristic is that it uses Flavin Mononucleotide (FMN) as a cofactor to perform its
function.

![D:/LYJ/2025-iGEM/李琦-构建可还原六价铬毒性的大肠杆菌工程菌株/最终实验结果汇总/质粒图谱/pSC101-chrR Map.pngpSC101-chrR Map](https://static.igem.wiki/teams/5737/engineer/13.webp)

**Figure 13. Plasmid Map of pSC101-chrR**

'
},{
"title": "Build 2",
"content": '

We constructed pSC101-ChrR through homologous
recombination. After bacterial culture, the pSC101 underwent double enzyme digestion, adding ApaI and PacI restriction
endonucleases that recognized their restriction sites, and now exists in a linearized plasmid form. The gel
electrophoresis diagram indicates that the linearized pSC101 is roughly around the value 10498 bp, as the band is
above the 8000 bp marker.

![descript](https://static.igem.wiki/teams/5737/engineer/14.webp)

**Figure 14. Gel Electrophoresis Diagram for Linearized pSC101**

Our desired gene in this experiment was ChrR, which
initially experienced Polymerase Chain Reaction (PCR) amplification to produce many copies of that gene. After PCR, we
dyed the desired genes with Loading Buffer, and, along with Small (100-2000bp) and Big (500-15000bp) Markers, we
inserted the fluids into a Gel Electrophoresis Machine. The glowing bands indicate that the ChrR DNA has been
successfully amplified, as its band is visualized around ~634 bp, indicating that it follows the expected size of the
gene. Thus, these successfully amplified genes will be used in homologous recombination.

![descript](https://static.igem.wiki/teams/5737/engineer/15.webp)

**Figure 15. Gel Electrophoresis Diagram for Gene ChrR**

Following this, gene fragment recovery was conducted
to reuse the genes for further testing. The ChrR gene was inserted into the linear pSC101 vector. Heat shock transfer
was employed to transfer pSC101-ChrR into EPI400 bacteria, which were later cultured. Following PCR amplification of
pSC101-ChrR, gel electrophoresis confirmed successful ChrR amplification, which indicates successful formation of
pSC101-ChrR. The reconstructed plasmid was sent to Tsingke, a genomic sequencing company, for results. Genomic
sequencing confirmed that our recombined plasmid contained the desired gene within multiple *E. coli* cell
colonies and confirmed prior estimates of ChrR.

![D:/LYJ/2025-iGEM/李琦-构建可还原六价铬毒性的大肠杆菌工程菌株/最终实验结果汇总/PCR扩增、验证、测序、菌落图/ChrR-组图_画板 1_画板 1.jpgChrR-组图_画板 1_画板 1](https://static.igem.wiki/teams/5737/engineer/16.webp)

**Figure 16. pSC101-ChrR Colony Monoclonal Identification Chart**

'
},{
"title": "Test 2",
"content": '

1. **Protein Expression**

We successfully transformed the pSC101-ChrR into*E. coli* BL21(DE3) using the heat shock transformation method. After roughly 15 hours, the resulting cell culture
was displayed as such:

![descript](https://static.igem.wiki/teams/5737/engineer/17.webp)

**Figure 17. Cell Culture Display of BL21(DE3) Bacteria with pSC101-ChrR Gene**

After inoculation and incubation of the bacteria, we
did another electrophoresis test to check if the plasmid was successfully transferred into the *E. coli*
BL21(DE3). The results show that it is around 634bp, matching the prior PCR bp length, confirming that we successfully
transferred pSC101-ChrR into BL21(DE3).

![descript](https://static.igem.wiki/teams/5737/engineer/18.webp)

**Figure 18. Gel Electrophoresis Diagram from the Colony PCR of Bacteria in an Agar Plate**

We cultivated more BL21(DE3) containing pSC101-ChrR
inside tubes until it reached OD600~0.6 for 4 hrs, 37ºC, 220 rpm. At varying rhamnose concentrations, we placed the
tubes in 16ºC, 20 hours, 220 rpm, for optimal gene expression and protein production. Following purification of crude
protein, we ran an SDS-PAGE to verify the protein expression. After checking, we found that the ChrR proteins are
around 24kDa, which infers the successful production of ChrR.

![](https://static.igem.wiki/teams/5737/engineer/19.webp)

**Figure 19. SDS-PAGE Diagram of ChrR Crude (Left Region) and Pure (Right Region)**

2. **Resistance to Chromium**

To test resistance to chromium, we took samples at
the end of each hour to put into tubes to do OD600 measurements. We measured at concentrations of 0, 10, 30, 50, 80,
and 100 mg/L at 37ºC, 220 rpm. pSC101-ChrR grows naturally from concentrations of 0-50 mg/L, with limited growth
exhibited at 80 mg/L. Growth was completely inhibited at 100 mg/L concentration.

The toxicity assessment experiments in this study
clearly demonstrate that as the Cr(VI) treatment concentration increases, a corresponding decrease in culture
turbidity (OD600) is observed. This directly quantifies the reduction in bacterial biomass and proliferation activity.
Through systematic testing, we determined the maximum tolerable concentration of Cr(VI) for this strain to be 80 mg/L.
Below this threshold, the strain exhibits growth but shows marked inhibition; when concentrations exceed this critical
value, growth is completely suppressed, and the culture becomes clear, indicating that cells cannot divide normally
and may even die.

![D:/LYJ/2025-iGEM/李琦-构建可还原六价铬毒性的大肠杆菌工程菌株/最终实验结果汇总/耐铬实验/pSC101-chrR不同Cr浓度处理.pngpSC101-chrR不同Cr浓度处理](https://static.igem.wiki/teams/5737/engineer/20.webp)

**Figure 20. Bacteria with pSC101-ChrR Gene Grown in Different Cr(VI) Concentrations, Image Taken After 6 Hours
of Growth**

The significant reduction in colony count with
increasing Cr(VI) concentration directly reflects the potent toxic effects of Cr(VI) on bacterial cells. The maximum
tolerable concentration of 80 mg/L indicates that below this threshold, the strain\'s detoxification mechanisms may
partially neutralize the oxidative toxicity of Cr(VI), sustaining basic survival. However, once concentrations exceed
this limit, severe oxidative stress leads to irreversible damage to cellular structures, metabolic dysfunction, and
ultimately cell death, preventing the formation of visible colonies on agar plates.

![D:/LYJ/2025-iGEM/李琦-构建可还原六价铬毒性的大肠杆菌工程菌株/最终实验结果汇总/耐铬实验/pSC101-chrR不同Cr浓度处理-菌落图.pngpSC101-chrR不同Cr浓度处理-菌落图](https://static.igem.wiki/teams/5737/engineer/21.webp)

**Figure 21. Bacteria with pSC101-ChrR Gene Grown in Different Cr(VI) Concentrations Spread on Agar Plate for
Growth Visualization**

The results of OD600 for pSC101-ChrR under each
concentration of Cr(VI) are shown. Analysis of the chart data reveals that pSC101-ChrR exhibits robust growth within
the concentration range of 0 to 80 mg/L, maintaining a stable growth rate with significant biomass accumulation.
However, when concentrations reached 100 mg/L or higher, the strain\'s growth was markedly inhibited, with a
significant decline in growth rate and eventual stabilization of biomass, indicating that this concentration
approached or reached its upper growth threshold.

![descript](https://static.igem.wiki/teams/5737/engineer/22.webp)

**Figure 22. Growth Curve Visualization of Bacteria with pSC101-ChrR from 0~6h, in Concentrations (A) 0 mg/L,
(B) 10 mg/L, (C) 30 mg/L, (D) 50 mg/L, (E) 80mg/L, (F) 100 mg/L**

3. **Quantification of Reduction**

As a general trend, ChrR was effective at decreasing
the amount of Cr(VI) concentration, with end concentrations being relatively similar. The graph indicates that there
is a generally decreasing trend for Cr(VI) concentration with the increase in the variable time. Moreover, the slope
of the line dropped at the fifth hour, displaying that the fifth hour is the optimum time for Cr(VI) degradation.

![D:/LYJ/2025-iGEM/李琦-构建可还原六价铬毒性的大肠杆菌工程菌株/最终实验结果汇总/铬降解实验/ChrR降解铬曲线.tifChrR降解铬曲线](https://static.igem.wiki/teams/5737/engineer/23.webp)

**Figure 23. Combined Graph of ChrR Protein\'s Cr(VI) Degradation Under Different Cr(VI) Concentrations**

'
},{
"title": "Learn 2",
"content": '

In this experiment, we tested Cr(VI) reduction and
growth a limited number of times; in the future, we can work towards running more experiments and validating this
information.

In the experiment, despite employing a method
involving three parallel replicate sampling tests, errors still exist in the actual testing process. This necessitates
multiple experiments to reduce such errors. Additionally, due to the complexity of actual samples, further
confirmation and repeated verification are required during field measurements.

'
}]
}, {
"title": "Part 3: pSC101-YieF",
"content": '',
"children": [{
"title": "Design 3",
"content": '

pSC101-YieF is composed of the
chromate-reducing-protein-producing gene ChrR combined with the plasmid backbone pSC101.

**pSC101** is a specialized and
low-copy-numbered cloning vector that has kanamycin resistance, lacks the oriT transfer origin, which prevents the
spreading of genes to other bacteria, a specific rhamnose-induced pathway, and a specialized optimal replicating range
at 37 °C.

YieF is a gene that is able to produce YieF, a soluble dimeric flavoprotein specialized in Cr(VI) degradation
(Ackerley), that is derived from the bacteria *E. coli MG1655*. One characteristic of YieF is that the protein
YieF is able to hold onto 4 electrons at a time, displaying a high efficiency.

![D:/LYJ/2025-iGEM/李琦-构建可还原六价铬毒性的大肠杆菌工程菌株/最终实验结果汇总/质粒图谱/pSC101-YieF Map.pngpSC101-YieF Map](https://static.igem.wiki/teams/5737/engineer/24.webp)

**Figure 24. Plasmid Map of pSC101-YieF**

'
},{
"title": "Build 3",
"content": '

The plasmid pSC101 was extracted from bacterial
culture, and underwent Double Enzyme Digestion by adding ApaI and PacI restriction endonucleases that recognized their
respective restriction sites. After finishing the plasmid mixture, we dyed the plasmid using Loading Buffer, along
with Big Markers (500-15000bp), and placed it in a gel electrophoresis machine. The gel electrophoresis diagram above
indicates that the linearized pSC101 is roughly around the value 10498 base pairs, as the band is above the 8000bp
marker.

![descript](https://static.igem.wiki/teams/5737/engineer/25.webp)

**Figure 25. Gel Electrophoresis Diagram for Linearized pSC101**

After PCR of YieF, gel electrophoresis revealed that
YieF\'s band was around 640 bp, indicating that it follows the expected size of the gene. Following this, gene fragment
recovery was conducted to reuse the genes for further testing. The ChrR gene was inserted into the linear pSC101
vector. Heat shock transfer was employed to transfer pSC101-YieF into EPI400 bacteria, which were later cultured.
Following PCR amplification of pSC101-YieF, gel electrophoresis confirmed successful YieF amplification, which
indicates successful formation of pSC101-YieF. The reconstructed plasmid was sent to Tsingke, a genomic sequencing
company, for results. Genomic sequencing confirmed that our recombined plasmid contained the desired gene within
multiple *E. coli* cell colonies and confirmed prior estimates of YieF.

![YieF-组图_画板 1](https://static.igem.wiki/teams/5737/engineer/26.webp)

**Figure 26. pSC101-YieF Colony Monoclonal Identification Chart**

'
},{
"title": "Test 3",
"content": '

We cultivated more BL21(DE3) containing pSC101-YieF
inside tubes until it reached OD600~0.6 for 4 hrs, 37ºC, 220 rpm. At varying rhamnose concentrations, we placed the
tubes in 16ºC, 20 hours, 220 rpm, for optimal gene expression and protein production. Following purification of crude
protein, we ran an SDS-PAGE to verify the protein expression. After checking, we found that the YieF proteins are
around 24kDa, which infers the successful production of YieF.

![](https://static.igem.wiki/teams/5737/engineer/27.webp)

**Figure 27. SDS-PAGE Diagram of YieF Crude (Left Region) and Pure (Right Region)**

**1. Resistance to Chromium**

To test resistance to chromium, we took samples at
the end of each hour to put into tubes to do OD600 measurements. We measured at concentrations of 0, 10, 30, 50, 80,
and 100 mg/L at 37ºC, 220 rpm. *pSC101-YieF* grows naturally from concentrations of 0-50 mg/L, with limited
growth exhibited at 80 mg/L. OD600 value of p*SC101-YieF* was the highest, demonstrating growth under stress.

The toxicity assessment experiments in this study
clearly demonstrate that as the Cr(VI) treatment concentration increases, a corresponding decrease in culture
turbidity (OD600) is observed. This directly quantifies the reduction in bacterial biomass and proliferation activity.
Through systematic testing, we determined the maximum tolerable concentration of Cr(VI) for this strain to be 80 mg/L.
Below this threshold, the strain exhibits growth but shows marked inhibition; when concentrations exceed this critical
value, growth is completely suppressed, and the culture becomes clear, indicating that cells cannot divide normally
and may even die.

![D:/LYJ/2025-iGEM/李琦-构建可还原六价铬毒性的大肠杆菌工程菌株/最终实验结果汇总/耐铬实验/pSC101-YieF不同Cr浓度处理.pngpSC101-YieF不同Cr浓度处理](https://static.igem.wiki/teams/5737/engineer/28.webp)

**Figure 28. Bacteria with pSC101-YieF Gene Grown in Different Cr(VI) Concentrations, Image Taken After 6 Hours
of Growth**

The significant reduction in colony count with
increasing Cr(VI) concentration directly reflects the potent toxic effects of Cr(VI) on bacterial cells. The maximum
tolerable concentration of 80 mg/L indicates that below this threshold, the strain\'s detoxification mechanisms may
partially neutralize the oxidative toxicity of Cr(VI), sustaining basic survival. However, once concentrations exceed
this limit, severe oxidative stress leads to irreversible damage to cellular structures, metabolic dysfunction, and
ultimately cell death, preventing the formation of visible colonies on agar plates.

![D:/LYJ/2025-iGEM/李琦-构建可还原六价铬毒性的大肠杆菌工程菌株/最终实验结果汇总/耐铬实验/pSC101-YieF不同Cr浓度处理菌落图.pngpSC101-YieF不同Cr浓度处理菌落图](https://static.igem.wiki/teams/5737/engineer/29.webp)

**Figure 29. Bacteria with pSC101-YieF Gene Grown in Different Cr(VI) Concentrations Spread on Agar Plate for
Growth Visualization**

The results of OD600 for pSC101-YieF under each
concentration of Cr(VI) are shown. Analysis of the chart data reveals that pSC101-YieF exhibits robust growth within
the concentration range of 0 to 80 mg/L, maintaining a stable growth rate with significant biomass accumulation.
However, when concentrations reached 100 mg/L or higher, the strain\'s growth was markedly inhibited, with a
significant decline in growth rate and eventual stabilization of biomass, indicating that this concentration
approached or reached its upper growth threshold.

![D:/LYJ/2025-iGEM/李琦-构建可还原六价铬毒性的大肠杆菌工程菌株/最终实验结果汇总/耐铬实验/yief组图_画板 1.tifyief组图_画板 1](https://static.igem.wiki/teams/5737/engineer/30.webp)

**Figure 30. Growth Curve Visualization of Bacteria with pSC101-YieF from 0~6h, in Concentrations (A) 0 mg/L,
(B) 10 mg/L, (C) 30 mg/L, (D) 50 mg/L, (E) 80mg/L, (F) 100 mg/L**

**2. Quantification of Reduction**

As a general trend, YieF was very effective at
reducing Cr(VI) quickly and efficiently, within the first 5 hours, most of the chromium had been reduced. YieF\'s
diagram shows a decreasing trend, where at the 5th hour, the rate of Cr(VI) degradation is significantly reduced. In
other words, the degradation rate of Cr(VI) after the 5th hour does not exhibit a significant difference from the rate
at the 12th hour. It is able to conclude that the 5th hour is the most optimum and the most efficient time for Cr(VI)
degradation.

![D:/LYJ/2025-iGEM/李琦-构建可还原六价铬毒性的大肠杆菌工程菌株/最终实验结果汇总/铬降解实验/YieF降解铬曲线.tifYieF降解铬曲线](https://static.igem.wiki/teams/5737/engineer/31.webp)

**Figure 31. Combined Graph of YieF Protein\'s Cr(VI) Degradation Under Different Cr(VI) Concentrations**

'
}, {
"title": "Learn 3",
"content": '

In this experiment, we only tested Cr(VI) reduction
and growth a limited number of times; in the future, we can work towards running more experiments and validating this
information. In the experiment, despite employing a method involving three parallel replicate sampling tests, errors
still exist in the actual testing process. This necessitates multiple experiments to reduce such errors. Additionally,
due to the complexity of actual samples, further confirmation and repeated verification are required during field
measurements.

'
}]
},{
"title": "Part 4: pSC101-ChrR-YieF",
"content": '',
"children": [{
"title": "Design 4",
"content": '

**pSC101-ChrR-YieF** is composed by the
Chromate-reducing-protein producing gene ChrR and a soluble dimeric flavoprotein specialized in Cr(VI) degredation
producing gene YieF combined with the plasmid backbone pSC101

**pSC101** is a specialized and
low-copy-numbered cloning vector that has kanamycin resistance, lacking the oriT transfer origin, which prevents the
spreading of genes to other bacteria, a specific rhamnose-induced pathway, and a specialized optimal replicating range
at 37℃ specifically.

**ChrR** is a gene that produces
**chromate-reducing flavoprotein** ChrR. It is derived from *Pseudomonas putida*. Its key
characteristic is that it uses Flavin Mononucleotide (FMN) as a cofactor to perform its function.

*YieF* is a gene that can produce YieF soluble dimeric flavoprotein specialized in Cr(VI) degradation
(Ackerley)-that is derived from the bacteria *E. coli MG1655*. One characteristic of YieF is that the protein
YieF is able to hold onto 4 electrons at a time, displaying a higher efficiency than ChrR, which can only handle 2
electrons at a time, as proven later in the experiment.

![D:/LYJ/2025-iGEM/李琦-构建可还原六价铬毒性的大肠杆菌工程菌株/最终实验结果汇总/质粒图谱/pSC101-chrR-Yief Map.pngpSC101-chrR-Yief Map](https://static.igem.wiki/teams/5737/engineer/32.webp)

**Figure 32. Plasmid Map of pSC101-ChrR-YieF**

'
},{
"title": "Build 4",
"content": '

The plasmid pSC101 was extracted from bacterial
culture, and underwent Double Enzyme Digestion by adding ApaI and PacI restriction endonucleases that recognized their
respective restriction sites. After finishing the plasmid mixture, we dyed the plasmid using Loading Buffer, along
with Big Markers (500-15000bp), and placed it in a gel electrophoresis machine. The gel electrophoresis diagram above
indicates that the linearized pSC101 is roughly around the value 10498 base pairs, as the band is above the 8000bp
marker.

![descript](https://static.igem.wiki/teams/5737/engineer/33.webp)

**Figure 33. Gel Electrophoresis Diagram for Linearized pSC101**

After PCR of ChrR, gel electrophoresis revealed that
ChrR\'s band was around 634 bp, indicating that it follows the expected size of the gene. Furthermore, YieF\'s band was
around 640 bp, also satisfying the expected range. Following this, gene fragment recovery was conducted to reuse the
genes for further testing. The ChrR and YieF genes were inserted into the linear pSC101 vector. Heat shock transfer
was employed to transfer pSC101-ChrR-YieF into EPI400 bacteria, which were later cultured. Following PCR amplification
of pSC101-ChrR-YieF, gel electrophoresis confirmed successful ChrR and YieF amplification, which indicates successful
formation of pSC101-ChrR-YieF. The reconstructed plasmid was sent to Tsingke, a genomic sequencing company, for
results. Genomic sequencing confirmed that our recombined plasmid contained the desired gene within multiple
*E. coli* cell colonies and confirmed prior estimates of ChrR and YieF.

![ChrR-yief-组图_画板 1](https://static.igem.wiki/teams/5737/engineer/34.webp)

**Figure 34. pSC101-ChrR Colony Monoclonal Identification Chart**

'
},{
"title": "Test 4",
"content": '

**1. Protein Expression**

We cultivated more BL21(DE3) containing
pSC101-ChrR-YieF inside tubes until it reached OD600~0.6 for 4 hrs, 37ºC, 220 rpm. At varying rhamnose concentrations,
we placed the tubes in 16ºC, 20 hours, 220 rpm, for optimal gene expression and protein production. Following
purification of crude protein, we ran an SDS-PAGE to verify the protein expression. After checking, we found that both
the ChrR proteins and YieF proteins are around 24kDa, which infers the successful production of ChrR and YieF.

![](https://static.igem.wiki/teams/5737/engineer/35.webp)

**Figure 35. SDS-PAGE Diagram of ChrR-YieF Crude (Left Region) and Pure (Right Region)**

**2. Resistance to Chromium**

To test resistance to chromium, we took samples at
the end of each hour to put into tubes to do OD600 measurements. We measured at concentrations of 0, 10, 30, 50, 80,
and 100 mg/L at 37ºC, 220 rpm. pSC101-ChrR-YieF grows naturally from concentrations of 0-50 mg/L, with limited growth
exhibited at 80 mg/L.

The toxicity assessment experiments in this study
clearly demonstrate that as the Cr(VI) treatment concentration increases, a corresponding decrease in culture
turbidity (OD600) is observed. This directly quantifies the reduction in bacterial biomass and proliferation activity.
Through systematic testing, we determined the maximum tolerable concentration of Cr(VI) for this strain to be 50 mg/L.
Below this threshold, the strain exhibits growth but shows marked inhibition; when concentrations exceed this critical
value, growth is completely suppressed, and the culture becomes clear, indicating that cells cannot divide normally
and may even die.

![D:/LYJ/2025-iGEM/李琦-构建可还原六价铬毒性的大肠杆菌工程菌株/最终实验结果汇总/耐铬实验/pSC101-chrR-YieF不同Cr浓度处理.pngpSC101-chrR-YieF不同Cr浓度处理](https://static.igem.wiki/teams/5737/engineer/36.webp)

**Figure 36. Bacteria with pSC101-ChrR-YieF Gene Grown in Different Cr(VI) Concentrations, Image Taken After 6
Hours of Growth**

The significant reduction in colony count with
increasing Cr(VI) concentration directly reflects the potent toxic effects of Cr(VI) on bacterial cells. The maximum
tolerable concentration of 50 mg/L indicates that below this threshold, the strain\'s detoxification mechanisms may
partially neutralize the oxidative toxicity of Cr(VI), sustaining basic survival. However, once concentrations exceed
this limit, severe oxidative stress leads to irreversible damage to cellular structures, metabolic dysfunction, and
ultimately cell death, preventing the formation of visible colonies on agar plates.

![D:/LYJ/2025-iGEM/李琦-构建可还原六价铬毒性的大肠杆菌工程菌株/最终实验结果汇总/耐铬实验/pSC101-chrR-YieF不同Cr浓度处理-菌落图.pngpSC101-chrR-YieF不同Cr浓度处理-菌落图](https://static.igem.wiki/teams/5737/engineer/37.webp)

**Figure 37. Bacteria with pSC101-ChrR-YieF Gene Grown in Different Cr(VI) Concentrations Spread on Agar Plate
for Growth Visualization**

The results of OD600 for pSC101-YieF under each
concentration of Cr(VI) are shown. Analysis of the chart data reveals that pSC101-YieF exhibits robust growth within
the concentration range of 0 to 50 mg/L, maintaining a stable growth rate with significant biomass accumulation.
However, when concentrations reached 80 mg/L or higher, the strain\'s growth was markedly inhibited, with a significant
decline in growth rate and eventual stabilization of biomass, indicating that this concentration approached or reached
its upper growth threshold.

![D:/LYJ/2025-iGEM/李琦-构建可还原六价铬毒性的大肠杆菌工程菌株/最终实验结果汇总/耐铬实验/chrr-yief组图_画板 1.tifchrr-yief组图_画板 1](https://static.igem.wiki/teams/5737/engineer/38.webp)

**Figure 38. Growth Curve Visualization of Bacteria with pSC101-ChrR-YieF from 0~6h, in Concentrations (A) 0
mg/L, (B) 10 mg/L, (C) 30 mg/L, (D) 50 mg/L, (E) 80mg/L, (F) 100 mg/L**

**3. Quantification of Reduction**

As a general trend, ChrR-YieF was effective at
decreasing the amount of Cr(VI) concentration, with end concentrations showing significant decreases. It has a
relatively constant slope, explaining its constant yet slow degradation rate.

![D:/LYJ/2025-iGEM/李琦-构建可还原六价铬毒性的大肠杆菌工程菌株/最终实验结果汇总/铬降解实验/ChrR-YieF降解铬曲线.tifChrR-YieF降解铬曲线](https://static.igem.wiki/teams/5737/engineer/39.webp)

**Figure 39. Combined Graph of ChrR-YieF Protein\'s Cr(VI) Degradation Under Different Cr(VI)
Concentrations**

'
},{
"title": "Learn 4",
"content": '

In this experiment, we only tested Cr(VI) reduction
and growth a limited number of times; in the future, we can work towards running more experiments and validating this
information.

In the experiment, despite employing a method
involving three parallel replicate sampling tests, errors still exist in the actual testing process. This necessitates
multiple experiments to reduce such errors. Additionally, due to the complexity of actual samples, further
confirmation and repeated verification are required during field measurements.

'
}]
}]%}
{% block title %}Engineering{% endblock %}
{% block lead %}

![](https://static.igem.wiki/teams/5737/banner/v1/wet-lab.webp)
![](https://static.igem.wiki/teams/5737/common/wetlab-1.webp)

WET LAB

ENGINEERING

{% endblock %}
{% block name %}wetlab{% endblock %}

{% extends "layout.html" %}
{% set table\_data = [{
"title": "Plasmid Construction",
"content": '
',
"children": [{
"title": "1.1 PCR Amplification of Target Genes",
"content": '

**Materials:**

| DNA(Chr-T7-amilCP,ChrR,YieF) | 2μL |
| --- | --- |
| Micropippetes and their tips | **\** |
| PCR tubes (Each DNA with 2 tubes) | 6 tubes |
| 2x T8 High Fidelity Master Mix | 25 μL |
| Forward Primer (amilCP\YieF\ChrR) | 2 μL (Tsingke） |
| Reverse Primer (amilCP\YieF\ChrR) | 2 μL (Tsingke） |
| ddH2O | 19 μL |

**Procedures:**

1. Gently Pipette DNA (Chr-T7-amilCP, ChrR, YieF) 2
μL, 25 μL 2xPCR Master Mix, 2 μL, 19 μL ddH2O into the 6 PCR tubes, all placed on ice.

2. Place tubes in the PCR machine.

3. Initial Denaturation: 95 ℃, 5 min.

4. Further Denaturation: 95 °C, 30 sec.

5. Annealing: 55 ℃, 30 sec.

6. Extension: 72 ℃, 1 min.

7. Final Extension: 72 ℃, 10 min.

8. Hold: ∞.

'
},{
"title": "1.2 Gel Electrophoresis of Target Genes",
"content": '

**Materials:**

| **Total** | **50mL** |
| --- | --- |
| 1xTAE | 50 mL |
| Agarose | 0.5g (Sangon) |
| Gel red | 3 μL (Easen) |

**Procedures:**

1. Measure about 50 mL of 1xTAE.

2. Measure 0.5g of Agarose.

3. Put into the microwave, and heat for about 4
minutes until everything has melted.

4. Pipette 3 μL of Gel red into the solution.

5. Make sure everything is distributed evenly.

6. Clean the mold and the mold piece thoroughly.

7. Insert the mold piece, and pour the solution into
the mold.

'
},{
"title": "1.3 Innoculation of Bacteria Culture",
"content": '

**Materials (Liquid\Solid):**

|  |  |
| --- | --- |
| Yeast Extract (Sangon） | 2.5g |
| NaCl (Sangon) | 5g |
| Tryptone (Sangon) | 5g |
| Digital balance, weighing paper, spoon | \ |
| 1 liter graduated cylinder | \ |
| ddH2O | 500 mL |
| **Solid: Agar (Sangon)** | **1.5g/100mL** |

**Procedures:**

**Liquid\Solid:**

1. Weigh 2.5g of yeast extract powder with weighing
paper and scale, and add it to the beaker.

2. Weigh 5g of NaCl (s) with weighing paper and
scale, and add to the beaker.

3. Weigh 5g of tryptone with weighing paper and
scale, and add it to the beaker.

4. Measure 500 mL of ddH2O with a
graduated cylinder, and add it to the beaker.

5. Weigh 5 portions of 1.5g agar powder, add to 5
beakers separately, and stir until all has dissolved(solid).

**Innoculation**

**Procedures:**

1. Disinfect both hands and supplies.

2. Pipette 5x5mL of LB liquid into each test tube.

3. Pipette 50 μL of *E. coli* solution (2
different types) into each test tube.

4. Pipette 5 μL of antibiotic into each test tube.

5. Cap test tubes and seal firmly.

6. Put in the machine, shake at about 37℃.

'
},{
"title":"1.4 Plasmid Extraction of pET28a & pSC101",
"content":'

**Materials:**

|  |  |
| --- | --- |
| Collection Tubes (Vazyme) | μLtramicro-volume spectrophotometer (ZuoFei) |
| Pipette | Ethanol (Shanghai Titan Scientific co., ltd) |
| Centrifuge (Hengnuo) | FastPure DNA Mini Columns (Vazyme) |
| Buffer p1/p2/p3 (Vazyme) | Columns (Beyotime) |
| Buffer pw1/pw2 (Vazyme) | ddH2O(Milli Q) |

**Procedures:**

1. Use pipette to get 2mL of pET28a (same for pSC101) bacterial suspension liquid, add into 2mL collection tube.
2. Put in centrifuge at 10000 rpm, for 1 min.
3. Bacteria form a pellet at the bottom of the tube, and dump the supernatant.
4. Add in 250 μL Buffer P1 (make sure Buffer P1 has RNase A), use pipette at smaller volume (eg, 200 μL) to resuspend
   bacteria back into the liquid (blow the pipette repeatedly towards the mixture to disperse the bacteria).
5. Add in 250 μL Buffer P2 (softly tilt the tube repeatedly upside down to mix).
   * Lyses bacterial cells, releasing the plasmid DNA, chromosomal DNA, proteins, etc
6. Add 350 μL Buffer P3 and tilt to mix; a white, flocculent precipitate should appear. P3 simultaneously rids the
   mixture of bubbles.

* Chromosomal DNA + proteins become the precipitate
* Plasmid DNA is soluble in the mixture

7. Put in centrifuge at 12000 rpm, for 10 min.
   * Precipitate pellets down while the plasmid DNA is still in the liquid mixture
8. Put FastPure DNA Mini Columns in a 2 mL collection tube. Add in the mixtures from Step 7 using a pipette (250 μL,
   smaller volume) into the FastPure DNA Mini Columns, avoiding aspirating the precipitate into the mixture. In the
   end, we combined 2 columns of liquid into one FastPure DNA Mini Column.
   * Plasmid binds in the silica membrane (filter) in the middle; everything else flows through
9. Put the FastPure DNA Mini Column in a centrifuge at 12000 rpm for 1 min. Some liquid should’ve dropped in the
   collection column below the FastPure DNA Mini Column; dump that liquid out and continue using the same collection
   tube.
10. Add in 500 μL of Buffer PW1, and centrifuge the mixture at 12000 rpm for 1 min. Dump the filtered liquid out and
    continue using the same collection tube.
    * PW1 denatures nucleases that could be in the mixture (that could destroy plasmid DNA)
11. Add in 600 μL of Buffer PW2 (ensure that 80mL of ethanol is added into the bottle of PW2 before usage), and
    centrifuge the mixture at 12000 rpm for 1 min. Dump the filtered liquid out and continue using the same collection
    tube.
    * Ethanol makes nucleic acids less soluble, so they bind to silica better; PW2 washes away other residues.
12. Repeat step 11.
13. Do not add any liquid into the FastPure DNA Mini
    Columns and use an empty collection tube. Centrifuge this at 12000 rpm for 1 min, to get some remaining liquid to
    the
    bottom of the collection tube. Dispose of this liquid and keep the FastPure DNA Mini Column.
    * Get rid of the remaining ethanol, since ethanol could disrupt reactions performed later on
14. Get another tube of 1.5mL volume. Put the FastPure DNA Mini Columns on top of this empty tube, and use a pipette
    to transfer 40 μL of ddH2O into the top of the FastPure DNA Mini Column. Make sure the ddH2O
    is transferred on top of the silica membrane portion of the FastPure DNA Mini Column.
    * To make sure pure plasmid DNA comes off into the column and into the 1.5 tube
15. Wait 2 min for it to settle.
16. Put the mixture in a centrifuge at 12000 rpm for 2 min.
17. Check the DNA concentration in the remaining solution by putting it in a Ulatramicro-volume spectrophotometer.
    Pipette onto the small hole, record the concentration (ng/ μL) and purity (A260 ratio).

'
},{
"title": "1.5 Gel Electrophoresis of Chr-T7-amilcp,
ChrR, and YieF",
"content":'

**Materials:**

|  |  |
| --- | --- |
| Marker, 100~2000 bp (Sangon Biotech) | Marker, 500~15000 bp (Sangon Biotech) |
| 1x TAE (Tris-Acetate-EDTA) buffer | 10x Loading Buffer (Beyotime) |
| Gel Imaging System(CLiN） | Gel Electrophoresis Machine (wix-midiDNA) |

**Procedures:**

1. Add 1x TAE until the water level in the gel electrophoresis machine.
2. In each individual tube of amilCP/ChrR/YieF, add 8μL of Loading Buffer to each.
3. Prepare 10 μL of 100~2000 bp Marker and 10μL of 500~15000 bp Marker.
4. Insert fluids (10μL) into the holes in the gel, run the gel electrophoresis at 180 volts for 20 minutes.
5. Bring the gel to the Gel Imaging System, under UV light, to see the results.

'
},{
"title": "1.6 Double Enzyme Digestion",
"content": '

**Materials:**

| pET28a plasmid | XhoI enzyme | BamHI enzyme |
| --- | --- | --- |
| pSC101 plasmid | APaI enzyme | PacI enzyme |
| 10x loading buffer | ddH2O (Milli Q) | Centrifuge (hengnuo mini) |

**Procedures:**

|  |  |
| --- | --- |
| **Total** | **20 μL** |
| 10x Buffer | 1 μL |
| BamHI enzyme | 1 μL |
| XhoI enzyme | 1 μL |
| pET28a plasmid\pSC101 plasmid | 10 μL |
| ddH2O | 7 μL |

Once the mixture is complete, place it in the
centrifuge for a bit (a couple of seconds), then into the PCR machine, where it can incubate at 37℃ for 20 min. It
will be cutting the plasmids and making them linear.

'
},{
"title": "1.7 Gene Fragment Recovery",
"content": '

**Materials:**

|  |  |  |  |
| --- | --- | --- | --- |
| Linear pSC101 & pET28a | Buffer GDP (Vazyme) | Buffer GW (Vazyme) | FastPure DNA Mini Columns (Vazyme) |
| Collection Tubes (Vazyme) | 1.5 mL Columns (Beyotime) | Gel Electrophoresis Machine (wix-midiDNA) | Gel Imaging System(CLiN) |
| Pipette | Centrifuge (Hengnuo) | Ultramicro-volume spectrophotometer (ZuoFei) | Water Bath (jinghong) |

**Procedures:**

1. Place fluids into holes in the gel for gel electrophoresis. Wait 10 min for gel electrophoresis (see second
   experiment steps 5 & 6 after inserting liquid). Check the nature of the gel after electrophoresis underneath
   the
   Gel Imaging System.
2. Take out a knife and carefully slice through the bands that are kept for purification (all bands except for the
   markers); the same slices (eg, A & A) are put in the same 2 mL collection tube.
3. Measure the mass of the gels after subtracting the tube + gel mass from the tube’s mass.
4. Add in equal quantities (500 μL) of Buffer GDP in all gel tubes.
5. Place gels in a water bath, wait for 7 min, though ensure that throughout the process, the gels are occasionally
   taken out to tilt upside down and mix slightly. A mix was done after the first 5 min and after the total 7 min had
   passed. Ensure that the gel is completely melted.
6. Attach FastPure DNA Mini Columns to 2 mL collection tubes, transfer 700 μL gel liquid into the FastPure DNA Mini
   Columns using a pipette.
7. Place in centrifuge at 12000 rpm for 60s. Discard the liquid in the collection tube after.
8. Transfer 300 μL Buffer GDP to FastPure DNA Mini Columns. Wait for 1 min. Then place in the centrifuge at 12000
   rpm
   for 60s once more. Discard the remaining liquid in the collection tube after.
9. Transfer 700 μL Buffer GW to FastPure DNA Mini Columns. Place in centrifuge at 12000 rpm for 60s. Then, discard
   the remaining liquid in the collection tube. Repeat this step twice.
10. Place the empty collection tube back below the FastPure DNA Mini Columns and add it to the centrifuge at 12000
    rpm
    for 2 min. Discard the remaining liquid.
11. Place the FastPure DNA Mini Columns on a 1.5mL Column. Add 20 μL of ddH2O to the center of the
    silicon
    membrane. Wait for 2 min. Then place in a centrifuge at 12000 rpm for 1 min.
12. Discard the FastPure DNA Mini Column, and place the resulting liquid on the Ultramicro-volume spectrophotometer
    to
    test the concentration of the linear pET28a/pSC101, as well as that of the ChrR, YieF, and amilCP.

'
},{
"title": "1.8 Homologous Recombination & *E. coli* Culturing",
"content":'

**Materials:**

| DH5α CompetentCells | EPI400 Super Competent Cells(Beyotime) | Linear pET28a&pSC101 | AmilCP/ChrR/YieF genes |
| --- | --- | --- | --- |
| LB (Lysogeny broth) | Pipette | Water Bath (jinghong) | Container of ice |
| Biosafety cabinets | Incubator (jinghong) | Centrifuge (Hengnuo) | Kanamycin antibiotics (50mg/mL) |

**Procedures 1 :**

**pET28a - Chr-T7-amilCP**

|  |  |
| --- | --- |
| **Total** | **10 μL** |
| 2x CE Mix | 5 μL |
| pET28a linear | 4 μL |
| Chr-T7-amilCP | 1 μL |

**pSC101 - ChrR**

|  |  |
| --- | --- |
| **Total** | **10 μL** |
| 2x CE Mix | 5 μL |
| pSC101 linear | 4 μL |
| ChrR | 1 μL |

**pSC101 - YieF**

|  |  |
| --- | --- |
| **Total** | **10 μL** |
| 2x CE Mix | 5 μL |
| pSC101 linear | 4 μL |
| YieF | 1 μL |

**pSC101 - ChrR-YieF**

|  |  |
| --- | --- |
| **Total** | **10 μL** |
| 2x CE Mix | 5 μL |
| pSC101 linear | 3 μL |
| ChrR | 1 μL |
| YieF | 1 μL |

Once all are ready, place in a water bath at 50℃
for
20 min, then put in 4℃.

**Procedures 2 :**

1. Take 10 μL of the combined mixture out, then transfer it into 100 μL *E. coli* Competent Cells. Place the
   mixture in ice for 20 min.
2. Transfer to a 42℃ water bath for 45s, immediately take it out and put it in ice for 2 min.
3. Add 800 μL of LB without antibiotics to the mixture.
4. Place the combined mixture into a centrifuge at 220 rpm, 37℃, for 1 hour, in order to let the bacteria revive.
5. Prepare another set of LB that contains 100μL of kanamycin(50mg/mL)→ LB is heated slightly because it gets
   conjugated and hardens into a jello-like solid form after a day. When cooled down to a temperature around 37℃, add
   kanamycin.
6. After an hour is up, place the bacteria mixture in a centrifuge at 5000 rpm for 2 min.
7. Take 100 μL of supernatant through a pipette (resuspend), extract 50 μL each of pET28a-chr-T7-amilCP (4 times),
   pSC101-ChrR (2 times), pSC101-YieF (2 times), and pSC101-ChrR-YieF (2 times), and spread the fluid (the strands of
   *E. coli*) throughout the entirety of the agar plate.
8. Place in an incubator at 37℃ for the whole night to culture *E. coli*.

'
},{
"title": "1.9 PCR Verification of Target Gene Presence
in Plasmids",
"content":'

**Materials:**

|  |  |
| --- | --- |
| **Total** | **20 μL** |
| 2x Mix | 10 μL |
| Primer-F | 1 μL |
| Primer-R | 1 μL |
| DNA single colony | \ |
| ddH2O | 8 μL |

**Procedures:**

1. Split groups of cultures by the target genes in
the plasmids they contain. Prepare for PCR separately.

2. Prepare a PCR test solution with the above
materials in one tube, and repeat for each culture. Add DNA, use a pipette to gently pick up one distinct cluster of
cells from a culture, and place them inside the tube.

'
},{
"title": "1.10 Gel Electrophoresis Verification of
Target Gene Size in Plasmids",
"content": '

**Materials:**

|  |  |
| --- | --- |
| 1xTAE buffer | 50mL |
| Agarose | 0.5g |
| GelRed | 3μL |

**Procedures:**

1. Measure 0.5g Agarose and place it into a 100 mL
conical flask.

2. Add 50mL 1xTAE buffer into the flask.

3. Stir thoroughly so no powdered Agarose is
visible.

4. Heat in microwave for around 3minutes until
Agarose is fully dissolved.

5. Add 3 μL of GelRed and mix until the solution is
uniform in color.

6. Pour the solution into the tray with the ladder,
and leave to cool.

7. Add the previously prepared PCR DNA into the
wells, wait for 20 min.

8. View results under UV light.

'
}]
},{
"title": "Tolerability testing",
"content": '
',
"children":[{
"title":"2.1 Chromium Resistance Testing",
"content":'

**Make the solution for the test:**each
concentration group for 0mg/L, 10mg/L, 30mg/L, 50mg/L, 80mg/L, 100mg/L of chromium makes three big test tubes of
solution, respectively, sampling them after each hour for a total of 6 hours.

**Making the Test Tube Solution:** add
30 mL of LB and 30 μL of Kana to every tube. Then, add 300 μL of pSC101-ChrR bacterium, pSC101-YieF bacterium, and
pSC101-ChrR-YieF bacterium in each tube, respectively. Label each tube with the bacteria gene and the concentration
of
chromium in the solution.

Lastly, add the corresponding concentration of
previously diluted chromium in each solution:

**0mg/L: no chromium added to the prior
solution.**

**10mg/L: add 300 μL of chromium to the prior
solution.**

**30mg/L: add 900 μL of chromium to the prior
solution.**

**50mg/L: add 1.5mL of chromium to the prior
solution.**

**80mg/L: add 2.4mL of chromium to the prior
solution.**

**100mg/L: add 3mL of chromium to the prior
solution.**

After each hour (0/1/2/3/4/5/6), participants of
each
group sample the solution by extracting 2ml from the test tube solution from each of the 3 test solutions, and
placing
it into three 2ml centrifuge tubes, respectively.

'
},{
"title": "2.2 Optical Density Test",
"content": '

**Materials:**

|  |  |  |
| --- | --- | --- |
| Labgic L-VM-C vibrator | SpectraMax i3x machines | pSC101-ChrR, pSC101-YieF, and pSC101-ChrR-YieF |
| 24-well plates | Pipette and tips | Sample tubes. |

**Procedure:**

1. Evenly distribute sample of pSC101-ChrR, pSC101-YieF, and pSC101-ChrR-YieF using a vibrator (Labgic L-VM-C) for
   a
   few seconds.
   * Samples were made of LB + kan + Cr(VI) at 0, 30, 50, 80, 100 mg/L concentrations collected every hour from
     0-6.
2. Using three 24-well plates (3 rows x8 columns), collect 3 rounds of 20 μL from the vial into the well for each
   hour of bacterial growth. This was repeated for every single concentration of Cr(VI).
   * The three microwell plates corresponded to different bacterium strains (pSC101-ChrR, pSC101-YieF, and
     pSC101-ChrR-YieF). Each row corresponded to an hour (0-6), and 3 samples were taken for each hour.
   * Rows 1-3, 5-7, and 9-11 represent pSC101-ChrR, pSC101-YieF, and pSC101-ChrR-YieF, respectively.
   * Columns represented hours from the top to the bottom (0-6)
3. OD600 was tested via SpectraMax’s i3x machine at 600 nm, to measure the optical density of samples, for 100-0
   Cr(VI) mg/L density. The entire plate was selected for reading.
4. A growth curve was graphed for each concentration
   and bacteria.
5. OD600 values for each Hour6 sample was graphed.

'
},{
"title":"2.3 Chromium-Treated Bacteria Growth Testing",
"content": '

**Procedure:**

1. Collect the Cr(VI) treated bacterium (which has
been incubated for 20 hours), and collect 20 μL to test if it can grow.

2. Pipette 20 μL of varying density Cr (VI) treated
bacteria.

3. Pippete into the middle of the LB Plate, spread
evenly.

4. Incubate at 37 ℃ overnight.

'
}]
}, {
"title": "Protein expression and purification",
"content": '',
"children":[{
"title": "3.1 Optimal Inducer Concentration Testing",
"content": '

**Materials:**

| **LB broth** | **L-rhamnosestocksolution(50mM)** | **Bacterial strains** |
| --- | --- | --- |
| Kanamycin solution (50 mg/mL) | Sterile pipettes | centrifuge tubes |
| ZHICHENG shaking incubator | sealing film (Amcor) | Elastic bands |

**Procedure:**

1. Preparation: Properly sterilize and take out 15 tubes, 5 different inducer concentrations for each bacterial
   strain. Label each according to plasmid and inducer concentration.
2. Prepare LB culture: Add 30 mL LB broth to all tubes.
3. Inoculation: For each tube, add 1.5 mL of the corresponding bacterial strain (5% inoculum). For all tubes, add
   30
   µL of Kanamycin solution.
4. Growth to logarithmic phase: Seal the tubes, bundle together using elastic bands, then incubate at 37 °C,
   shaking
   at 220 rpm, until OD₆₀₀ ≈ 0.6 (~4 hours).\
5. Prepare the five groups from each of the three bacterial strains. Add L-rhamnose (50 mM stock) to reach final
   concentrations in 30 mL cultures as follows (C₁V₁ = C₂V₂):

**0.10 mM: add 60 µL stock**

**0.25 mM: add 150 µL stock**

**0.50 mM: add 300 µL stock**

**0.75 mM: add 450 µL stock**

**1.00 mM: add 600 µL stock**

- Induction phase: After adding the inducer, first
  seal using the sealing film, bundle together using elastic bands, then shift the incubator to 16
  °C, continue shaking at 220 rpm for 20 hours.

'
},{
"title": "3.2 Protein Purification",
"content": '

**3.2.1 Lysis and Purification**

**Procedure:**

1. Centrifuge bacterial suspension at 5400 rpm for 15 minutes at 4℃. (Eppendorf centrifuge 5810 R, make sure to
   balance the centrifuge.)
2. Decant the supernatant and pipette out residual liquid until only the pellet is left. (Make sure not to disrupt
   the cell pellet, and use the pipette volume according to the residual liquid.)
3. Add 5 mL of Beyotime Denaturing Lysis Buffer to the pellet. Mix evenly to avoid clumping or condensate
   formation.
   Use the Labgic L-VM-C vibrator in order to mix samples.
4. Add 50 μL of lysosome (enzyme stock,60 mg stored at 4℃) to the sample.
5. Incubate samples in ice bath or on ice for 20 minutes, or until sonicator is ready.
6. Use the BioSafer Sonicator to disrupt the cell and complete cell lysis. Keep samples on ice to prevent heat
   denaturation. Set settings to 50 W, 10 minutes, 3 seconds off, 3 seconds on, 50 percent yield. (Ensure that when
   undergoing sonification, the sample remains submerged in the ice bucket without too much ice melting, and the
   sample
   is in the center, with the probe not touching the sides of the tube. Remove samples if it starts bubbling or a
   muffled sound emerges instead of a sharp one.)
7. Bring samples to centrifuge for 10 minutes, 4000 rpm. After centrifugation, pipette 2 mL into a 2 mL tube (crude
   protein)
8. Pipette remaining liquid into 15 mL tubes for purification. Add 1mL Ni-NTA (His-tag purification resin) into the
   tubes. ( 6x His-tag on the end of ChrR and YieF to mark the protein, and Ni-NTA combines with 6x His-tag and keeps
   the product to ensure purification.)
9. Take a 15 mL tube and put it in a vortex mixer at 4℃ for 2 hours.

**3.2.2 Extraction**

**Procedure:**

1. Label Affinity columns with protein name (ChrR, YieF, ChrR-YieF) and its concentration of rhamnose (0.25, 0.5,
   0.75, 1 mM), and remove the bottom cap of the column. Slowly pipette all the protein-Ni-NTA solution into the
   Affinity column. Add 1 mL of non-denaturing Washing Buffer to the column dropwise. Apply 5 times to remove the
   unwanted proteins
2. Label microcentrifuge tubes with the protein’s name and rhamnose concentration, preparing two tubes for each
   protein-concentration pair. Add 0.5 mL of non-denaturing elution buffer 6 times to each of the microcentrifuge
   tubes. Collect the eluate into the labeled tube.
3. Put into the Microvolume Spectrophotometer (Dade Chuangtong (Shanghai) Technology Co., Ltd.) and read results.

'
},{
"title": "3.3 SDS-PAGE Testing",
"content": '

**Materials:**

| Gel casting mold | 2x Lower gel solution | 2x Upper gel solution |
| --- | --- | --- |
| 1.00 mm glass plates | 2x Lower gel buffer | 2x Coloured upper gel buffer |
| Comb | sterile tips | Modified polymerization initiator |
| Pipettes | mixing cups | isopropanol |

**Procedure:**

1. Assemble mold: Wash the mold with water and
   securely (with no leakage) set up the gel casting mold with clean 1.00 mm glass plates.
2. Prepare resolving (lower) gel: Mix 2.7 mL 2x
   lower
   gel solution with 2.7 mL 2x lower gel buffer in a cup. Add 55 µL modified polymerization initiator, mix gently,
   avoiding bubbles.
3. Pour resolving gel: Fill the space between
   plates
   until 1.5 cm from the top edge. Overlay gently with isopropanol to create a flat surface and prevent oxygen
   inhibition.
4. Leave at 25 °C for 6–10 minutes until a boundary
   forms, indicating solidification.
5. Prepare stacking (upper) gel: Remove the overlay carefully. In a new mixing cup, mix 0.75 mL 2X upper gel
   solution
   with 0.75 mL 2X coloured upper gel buffer. Add 15 µL modified polymerization initiator, mix gently, and avoid
   bubbles.
6. Pour stacking gel: Add the upper gel solution on top of the polymerized resolving gel until flush with the top
   of
   the glass plates.
7. Insert comb: Slowly insert the comb to form wells, taking care to avoid trapping bubbles. Leave at room
   temperature for 10-15 minutes.
8. Prepare wells: After gel solidification, carefully remove the comb. Wash wells with electrophoresis buffer using
   a
   pipette tip. The gel is now ready for protein loading and electrophoresis.

'
}]
}, {
"title": "Chromium degradation experiment",
"content": '',
"children":[{
"title": "4.1 Chromium degradation Test",
"content": '

**Procedure:**

1. Label each 50 mL centrifuge tube by chromium
concentration (10, 30, 50, 80, 100mg/L); hours of sampling (0, 3, 6, 16); and Protein name(ChrR, YieF, and
ChrR-YieF).

* [For one concentration ex:100mg/L Cr(VI), there is one tube of 0hr, 3hr, 6hr, 16hr respectively]

2.Add 10mL of Sangon Biotech 1x PBS(with 0.05%
Tween-20) Solution. Then, add 1mL of protein (ChrR, YieF, and ChrR-YieF) in each tube, respectively.

3. Add the corresponding concentration of
previously
diluted chromium in each solution:

1**0mg/L: add 10 μL of chromium to the prior
solution.**

**30mg/L: add 30 μL of chromium to the prior
solution.**

**50mg/L: add 50ml of chromium to the prior
solution.**

**80mg/L: add 80ml of chromium to the prior
solution.**

**100mg/L: add 100 μL of chromium to the
prior
solution.**

'
},{
"title": "4.2 Protein Transferring Chromium Rate
Test",
"content": '

**Materials:**

|  |  |  |  |
| --- | --- | --- | --- |
| Protein solutions of ChrR, YieF, and ChrR-YieF | Reagent 1 (SangonBiotech) | Cr(VI) reagent 2 (SangonBiotech) | 1.5mL centrifuge tubes |
| ddH2O | 96 Well and 8-Well Strips ELISA Plates (Sangon Biotech) | calibrator (Sangon Biotech) | Micropipette and its tips |

**Procedure:**

1. Use 0 h, 5 h, and 12 h as our target times and gather 17 1.5mL-centrifuge tubes per protein (ChrR, YieF, and
   ChrR
   & YieF)for the experiment (5 different concentrations of Cr VI, one standard tube or calibrator (Sangon), and
   one reagent blank containing ddH2O).
2. Take out 200 μL from each protein solution under different concentrations of Cr(VI) and transfer it into the
   1.5mL
   centrifuge tube.
3. Take out 200 μL of the calibrator solution (Sangon Biotech) and place it into the calibrator tube, and transfer
   200 μL of ddH2O into the reagent blank tube.
4. Add 10 μL reagents 1 and 2 (Sangon Biotech) to all tubes, place all 17 tubes onto the rack, and put the rack
   inside the incubator at 37 ℃ for 10 minutes.
5. Transfer 100 μL of the incubated protein solution into the microplate/ELISA plate twice per solution.
6. Transfer 100 μL of calibrator solution (Sangon Biotech) and ddH2O to the last row of the plate.
7. Place the plate into the microplate reader (Molecular Devices).

'
}]
},{
"title": "Chromium Detection experiment",
"content": '
',
"children":[{
"title": "5.1 Preparing Cultures Under Different
Chromium Concentrations",
"content": '

**Materials:**

|  |  |  |
| --- | --- | --- |
| LB broth: 30 mL | Kanamycin solution: 30µL | Chromium stock solution: 1000 mg/L |
| Bacterial strain | Sterile pipettes | 12mL Culture tubes |
| ZHICHENG shaking incubator | Sealing film (Amcor) | Elastic bands |

**Procedure:**

1. Preparation: Properly sterilize and take out 5 tubes, labelling each with the corresponding plasmid and chromium
   concentration.
2. Prepare cultures: Add (pipette) 5 mL LB broth into all tubes.
3. Add Antibiotic: Add (pipette) 5 µL per tube.
4. Inoculate bacteria: Add 50 µL of pET-Chr-T7-amilCP culture to each tube.
5. Add chromium stock: Using C₁V₁ = C₂V₂, add the volumes of 1000 mg/L chromium stock to achieve final
   concentrations
   of 0, 10, 30, 50, 80, and 100 mg/L.
6. Incubate: Seal the tubes using sealing film, bundle using elastic bands, then incubate overnight at 37 °C, 220
   rpm.

'},{
"title": "5.2 Bacterial Plating",
"content": '

**Materials:**

|  |  |  |
| --- | --- | --- |
| Bacterial Agar Plates | pET-Chr-T7-amilcp in different Cr(VI) concentrations | Mircopipettes |
| L-shaped spreader | Parafilm |  |

**Procedure:**

1. Prepare 12 agar plates (two each for 0, 10, 30, 50, 80, and 100mg/L), label the backside of the agar plates with
   different Chromium concentrations.
2. Place 6 of the tubes for spreading the bacteria in agar plates, and the remaining ones are used for centrifuging
   purposes later.
3. Take out 10 μL of the bacterial solution and eject 10 μL at the center of the agar plate with a pipette.
4. Use a L-shaped spreader to spread the solution evenly throughout the agar plate
5. Close the lid and seal the agar plate with parafilm. Close the cap for the 6 test tubes containing
   pET-Chr-T7-amilcp under different Cr(VI) concentrations.
6. Place the 12 plates into the constant temperature incubator at 37 ℃.

'
}]
}]%}
{% block title %}Experiments{% endblock %}
{% block lead %}

![](https://static.igem.wiki/teams/5737/banner/v1/wet-lab.webp)
![](https://static.igem.wiki/teams/5737/common/wetlab-1.webp)

WET LAB

EXPERIMENTS

{% endblock %}
{% block name %}wetlab{% endblock %}

{% extends "layout.html" %}
{% set table\_data = [{
"title": "Experiment Overview",
"content": '

**Heavily contaminated effluent from industry
leftovers threatens both human and environmental health. Among the toxic carcinogens is hexavalent chromium [Cr
(VI)], a highly toxic metallic element.** It is highly soluble in water across a wide range of pH conditions,
granting it the mobility to easily contaminate water sources and soil. Hexavalent chromium may be reduced to a less
toxic trivalent chromium [Cr (III)], where it isn’t as toxic or soluble. Accordingly, the two crucial steps to reduce
chromium contamination are chromium detection and chromium degradation. As a result, one detection plasmid
(pET28a-Chr-T7-amilCP) and three degradation plasmids (pSC101-ChrR, pSC101-YieF, and pSC101-ChrR-YieF) were designed,
built, tested, and then reflected.

The figure below illustrates the overall design and
goals of our experiment.

![实验流程图](https://static.igem.wiki/teams/5737/result/1.webp)

**Figure 1 Experimental Technology Roadmap**

'
},{
"title": "Chromium detection system",
"content": '',
"children": [{
"title": "1.1 Experimental Design and Mechanism Introduction",
"content": '

To create a chromium detection system, we constructed
pET28a-Chr-T7-amilcp, which expresses a blue color when chromium is detected. pET28a-chr-T7-amilCP is composed of four
parts: the plasmid backbone pET28a, chromium-biosensor regulatory module chr, a strong promoter and signal amplifier
T7, and a blue pigment protein amilCP.

**pET28a** is the widely used plasmid in
the pET (Plasmid for *E. coli* T7 expression) series, containing multiple features that aid the expression of
recombinant proteins in *E. coli*. The pET28a vector contains the selective marker kan, allowing the vector and
thus the bacteria to be resistant to the antibiotic kanamycin, providing selective pressure, and allowing only
bacteria with said selective marker to survive on kanamycin plates. Furthermore, pET28a encodes 6x histidine tags on
the gene, which can bind strongly to either Nickel (Ni2+) or Cobalt (Co2+). Additionally, the
gene contains multiple cloning sites with numerous restriction sites, allowing flexible insertion of genes of
interest. The lac operator sequence sits close to the T7 promoter, preventing leaky expression from occurring, thus
indicating the strong efficacy of pET28a as a vector.

**Chr** is a regulatory module that
controls the transcription of downstream genes.

![descript](https://static.igem.wiki/teams/5737/result/2.webp)

**Figure 2. chrB Gene Without Cr(VI) Present, Focusing On: p\_chr Operator, chrB Gene, and T7 Promoter in the
Regulatory Module chr**

Chr is composed of a p\_chr promoter+operator, which
recruits T7-RNA-polymerase that is vital for transcription to begin when activated, and a consistently expressed
repressor-producing gene, chrB, that codes for the repressor ChrB to inactivate the p\_chr operator when Cr(VI) is not
present.

![descript](https://static.igem.wiki/teams/5737/result/3.webp)

**Figure 3. chrB Gene with Cr(VI) Present, Focusing On: p\_chr Operator, chrB Gene, and T7 Promoter in the
Regulatory Module chr**

Repressor ChrB protein holds high affinity for the
promoter+operator sequence p\_chr, and binds to it when Cr(VI) is not prevalent. This binding inactivates p\_chr and its
ability to recruit **T7-RNA polymerase**(**T7-RNAP**). As only T7-RNAP could bind to the T7
promoter, the lack of T7-RNAP suppresses transcription of downstream genes. The allosteric property of ChrB repressor
protein is activated when it binds to Cr(VI) ions(CrO42-,
Cr2O74-), which induces a conformational change that causes Cr(VI)-bound ChrB\'s
dissociation from the operator. This derepression of the operator allows the initiation of transcription for
downstream genes when T7-RNAP is recruited and binds to the T7 promoter.

The **T7 promoter** is a strong promoter
sequence that is **only** recognized by T7-RNAP and is not recognized by the bacterial host\'s own RNAP.
As an extremely strong amplifier, the T7 promoter can cause target genes to be transcribed so efficiently that they
comprise up to 30% of the total cellular mRNA within a short period. Consequently, the expressed protein can account
for more than 50% of the total cellular protein after only a few hours of induction.

The **amilCP gene** came from***Acropora millepora,*** and encodes a chromoprotein called amilCP. When the gene is expressed, the
resulting protein is naturally blue-purple without needing external aid for activation, producing a direct and visible
output.

![D:/LYJ/2025-iGEM/李琦-构建可还原六价铬毒性的大肠杆菌工程菌株/最终实验结果汇总/质粒图谱/pET28a-chr-T7-amilCp Map.pngpET28a-chr-T7-amilCp Map](https://static.igem.wiki/teams/5737/result/4.webp)

**Figure 4. Plasmid Map of pET28a-chr-T7-amilCp**

Our purpose for pET28a-chr-T7-amilCP is to create a
highly sensitive, visible blue biosensor that can tell us the presence of Cr(VI) in the water.

**Off stage:**

**The chrB** gene is consistently
expressed and continuously produces the ChrB repressor protein. The ChrB repressor binds specifically to the p\_chr
promoter+operon, which inactivates p\_chr and its ability to recruit T7 RNAP for transcription. Only T7-RNAP could bind
to the T7 promoter to cause transcription to happen, so without T7-RNAP, there will not be transcription.

**![descript](https://static.igem.wiki/teams/5737/result/5.webp)**

**Figure 5. chr-T7-amilCP Pathway Under No
Cr(VI) Presence**

**Inducer intake Cr(VI)：**

When Cr(VI) is added to the solution with bacteria,
the bacteria take up the molecule. Hexavalent chromium enters bacterial cells through specific transport proteins on
the bacterial cell membrane, using "disguised" entry via the active transport systems of essential nutrients (such as
sulfate and phosphate). In aqueous solutions, hexavalent chromium often exists in the form of chromate (CrO₄²⁻) or
dichromate (Cr₂O₇²⁻); these chemical structures are highly similar to that of essential bacterial sulfate (SO₄²⁻). The
transport proteins on the bacterial cell membrane responsible for sulfate uptake (such as sulfate permeases) cannot
effectively distinguish between the two and could mistakenly identify the oxyanions of hexavalent chromium as sulfate,
which leads to the bacteria actively transporting (energy-dependent) Cr(VI) into the bacterial cell\'s cytoplasm,
coming into contact with the bacteria\'s plasmid.

**Cr(VI) induces conformational change of
repressor:**

After entering the cell, Cr(VI) binds to the ChrB
repressor protein. This binding changes the 3D shape (conformation) of the repressor, making it no longer able to bind
tightly and hence fall off from the operator.

**![descript](https://static.igem.wiki/teams/5737/result/6.webp)**

**Figure 6. chr-T7-amilCP Pathway with Cr(VI)
Present**

**T7-RNAP recruitment and transcription of the
gene:**

With the repressor gone, p\_chr is activated, which
recruits T7-RNAP. T7-RNAP binds to the T7 promoter, which greatly amplifies the signal. An abundant amount of mRNA is
made, which leads to the subsequent mass production of amilCP protein when a ribosome binds to the RBS and translates
the mRNA.

'
},{
"title": "1.2 Plasmid Construction for Detection
Systems",
"content": '

**1.2.1 pET28a Vector Extraction &
Linearization**

The plasmid **pET28a** was extracted
from bacterial culture, underwent Double Enzyme Digestion by adding XhoI and BamHI restriction endonucleases that
recognized their respective restriction sites, and now remains as linearized plasmid DNA. We then used an
Ultramicro-volume spectrophotometer (ZuoFei) to test the plasmid DNA\'s concentration (in ng/μL) and purity (in the
A260/A280 ratio, or the sample\'s UV light absorbance rate at 260nm divided by that value at 280nm).

![descript](https://static.igem.wiki/teams/5737/result/7.webp)

**Table 1. Concentration and Purity of pET28a
Plasmid**

Through repeated testing of 7 batches of plasmid DNA,
we chose two batches with relatively high concentration and purity, being batches 1 and 3. The concentration of these
two batches was respectively nearly double and triple that of the other batches, indicating their significantly higher
concentration, which will be more ideal for the homologous recombination of our desired genes. Since DNA absorbs UV
light maximally at 260nm, while proteins absorb UV light maximally at 280nm, pure DNA tends to have a ratio value of
around 1.8, while pure protein tends to have a ratio of 0.6. Thus, a far lower value may indicate protein
contamination, and a far higher value may indicate RNA contamination, or other impurities in general. While the
batches that we chose did not have the most ideal purity, we prioritized the higher concentration because the plasmid
is still required for downstream application, especially in homologous recombination, which requires enough template
DNA to ensure multiple efficient reactions. The A260/A280 values were not substantially far from 1.8, and were still
relatively closer to 1.8 (0.1~0.2 difference) compared to 0.6 (1.0~1.1 difference).

After finishing the plasmid mixture, we dyed the
plasmid using Loading Buffer, along with Big Markers (500-15000bp), and placed it in a gel electrophoresis machine.

![D:/LYJ/2025-iGEM/李琦-构建可还原六价铬毒性的大肠杆菌工程菌株/最终实验结果汇总/PCR扩增、验证、测序、菌落图/pET28a Linear 双酶切图.jpgpET28a Linear 双酶切图](https://static.igem.wiki/teams/5737/result/8.webp)

**Figure 7. Gel Electrophoresis Diagram for Linearized pET28a**

Figure 7 indicates a gel electrophoresis diagram of
the linearized pET28a, which is roughly around 5369 bp, as the glowing band is around the 6000 bp marker. From the
diagram above, the intensity and brightness of the pET28a band are relatively low, even in comparison with the marker.
However, its concentration remains sufficient to ensure the normal progression of subsequent experiments.

**1.2.2 Chr-T7-amilCP PCR Amplification**

Our desired gene in this experiment was
Chr-T7-amilCP, which initially experienced Polymerase Chain Reaction (PCR) amplification to produce many copies of
that gene. After PCR, we dyed the desired genes with Loading Buffer, and, along with Small (100-2000bp) and Big
(500-15000bp) Markers, we inserted the fluids into a Gel Electrophoresis Machine.

![D:/LYJ/2025-iGEM/李琦-构建可还原六价铬毒性的大肠杆菌工程菌株/最终实验结果汇总/PCR扩增、验证、测序、菌落图/Chr-T7-amilCP扩增图.jpgChr-T7-amilCP扩增图](https://static.igem.wiki/teams/5737/result/9.webp)

**Figure 8. Gel Electrophoresis Diagram for Gene Chr-T7-amilCP**

In Figure 8, the glowing bands indicate that the
*Chr-T7-amilCP* DNA has been successfully amplified, as its band is visualized around 1846 bp, indicating that
it follows the expected size of the gene. Thus, these successfully amplified genes will be used in homologous
recombination.

**1.2.3 Homologous Recombination and Heat Shock
Transfer**

We then set up a reaction mixture for inserting our
desired gene into the linear pET28a vector. Based on previous research, we realized that adding a small amount of
*E. coli* competent cells, such as DH5α cells, along with the plasmid and desired gene can serve as an in vivo
homologous recombination method [5]. Here, we chose DH5α cells specifically due to their features, including the recA1
mutation, which minimizes the risk of plasmid rearrangements or deletions during propagation, allowing the inserted
fragments to have high stability. Furthermore, it also possesses the endA1 mutation, which prevents the degradation of
plasmid DNA, thereby improving the yield and quality of DNA. Additionally, it contains another genetic modification
that allows for blue/white screening, meaning easier identification of recombinant clones.

We chose to add kanamycin to the LB mixture because
we were worried that the other bacteria or unwanted colonies would contaminate the rest of the bacteria. After roughly
15 hours, the resulting cell culture was displayed as such:

![descript](https://static.igem.wiki/teams/5737/result/10.webp)

**Figure 9. Cell Culture Display of DH5α Bacteria with pET28a-Chr-T7-amilCP Gene**

The remaining bacteria growing on this cell culture
should contain the plasmid and genomic sequences that we desire. However, further verification is still needed, as
there is a chance that other bacteria could have spontaneously mutated to develop resistance to kanamycin, though the
probability is low. Thus, we continued with more verification testing below.

**1.2.4 Colony PCR Identification**

Five colonies from the agar plate were chosen, and we
used our pipette to dab a little bit of the colony and place it into a PCR mix, along with primers (F, forward; and R,
reverse) that define the amplification region. This mixture is then placed into a PCR machine, and the final products
are loaded into an agarose gel for gel electrophoresis. The previous gel electrophoresis used to confirm successful
Chr-T7-amilCP amplification verifies that the gene should be around 1846 bp. The glowing band results indicated below
show roughly 1846 bp as well, indicating successful insertion of the gene into pET28a, and the successful formation of
pET28a-Chr-T7-amilCP.

![D:/LYJ/2025-iGEM/李琦-构建可还原六价铬毒性的大肠杆菌工程菌株/最终实验结果汇总/PCR扩增、验证、测序、菌落图/pET28a-T7-amilCP in DH5a验证图.pngpET28a-T7-amilCP in DH5a验证图](https://static.igem.wiki/teams/5737/result/11.webp)

**Figure 10. Gel Electrophoresis Diagram from the Colony PCR of Bacteria in an Agar Plate**

**1.2.5 Gene Sequencing**

The extracted, recombined plasmid, along with the F
and R primers, was then sent to a genomic sequence testing company, Tsingke. The results we received are displayed as
such:

![D:/LYJ/2025-iGEM/李琦-构建可还原六价铬毒性的大肠杆菌工程菌株/最终实验结果汇总/PCR扩增、验证、测序、菌落图/pET28a-chr-T7-amilCp测序.pngpET28a-chr-T7-amilCp测序](https://static.igem.wiki/teams/5737/result/12.webp)

**Figure 11. Gene Sequencing of pET28a-Chr-T7-amilCP**

After gene sequencing, the desired gene has once
again been verified to be around 1846 bp, matching the results from the colony PCR gel electrophoresis as well as the
initial gel electrophoresis that confirms gene PCR amplification. Thus, through these two instances of verification,
we successfully proved that our recombined plasmid contains our desired gene, and that said recombined plasmid exists
in multiple *E. coli* cell colonies.

'
},{
"title": "1.3 Chromium Sensor Validation",
"content": '

**1.3.1 Heat Shock Transformation of
pET28a-chr-T7-amilCP into *E. coli* BL21(DE3)**

BL21(DE3) is a form of B strain *E. coli* with
a mutation on the lon and ompT genes. Removing the presence of these two proteins allows for increased protein
expression. We successfully transformed the pET28a-chr-T7-amilCP into *E. coli* BL21(DE3) using the heat shock
transformation method. After roughly 15 hours, the resulting cell culture was displayed as such:

![descript](https://static.igem.wiki/teams/5737/result/13.webp)

**Figure 12. Cell Culture Display of BL21(DE3) Bacteria with pET28a-Chr-T7-amilCP Gene**

After inoculation and incubation of the bacteria, we
did another Colony PCR test to check if the plasmid was successfully transferred into the competent cell. The results
show that it is around 1846bp, matching the prior PCR bp length, confirming that we successfully transferred
pET28a-chr-T7-amilCP into BL21(DE3).

![descript](https://static.igem.wiki/teams/5737/result/14.webp)

**Figure 13. Gel Electrophoresis Diagram from the Colony PCR of Bacteria in an Agar Plate**

**1.3.2 Cr(VI) Detection Biosensor
Analysis**

We test the effectiveness of pET28a-chr-T7-amilCP in
detecting Chromium VI at different concentrations ( 0, 10, 50, 80, 100 mg/L ). The results show that the blue color
indicates that amilCP protein is present, with the higher intensity of blue(amilCP) prevalent, which indicates the
higher chromium concentration in the solution. However, if the levels of Cr(VI) concentration in the solution are too
high, the bacteria may die and cannot express the *amilCP* gene, leading to the absence of amilCP protein,
which leads to the absence of blue color. 30mg/L is discarded due to some practical errors, which lead to some
unreliable results.

![D:/LYJ/2025-iGEM/李琦-构建可还原六价铬毒性的大肠杆菌工程菌株/最终实验结果汇总/铬检测实验/0、10、50浓度铬颜色变化.png0、10、50浓度铬颜色变化](https://static.igem.wiki/teams/5737/result/15.webp)

**Figure 14. pET28a-chr-T7-amilCP Turning Blue with Cr(VI) Presence**

At the same time, we checked the tubes to see if they
contained the color blue because of amilCP\'s prevalence. We transferred the different concentrations of
Cr(VI)-pET28a-chr-T7-amilCP solution onto disks and observed their color more closely.

![D:/LYJ/2025-iGEM/李琦-构建可还原六价铬毒性的大肠杆菌工程菌株/最终实验结果汇总/铬检测实验/0、10、50浓度铬颜色变化菌落图.png0、10、50浓度铬颜色变化菌落图](https://static.igem.wiki/teams/5737/result/16.webp)

**Figure 15. Cell Culture Display of Bacterial Strains that Expressed amilCP Protein**

The results of the petri-dishes match our results of
the tubes: the higher the chromium concentration, the higher the intensity of blue.

'
}]
}, {
"title": "Chromium degradation system",
"content": '',
"children": [{
"title": "2.1 Design Mechanism",
"content": '

To create a chromium degradation system, we
constructed pSC101-ChrR, pSC101-YieF, and pSC101-ChrR-YieF, the three target plasmid recombinants. pSC101 indicates
the plasmid series. Chromate reductase ChrR (ChrR) contains the ChrR protein coding gene; chromate reductase YieF
(YieF) contains the YieF protein coding gene; and ChrR-YieF contains both YieF and ChrR protein coding genes. Both
ChrR and YieF proteins catalyze the reduction of chromium VI to the less toxic chromium III. pSC101 is an ideal vector
to recombine with the gene ChrR since ChrR is toxicity-sensitive, so high expression of ChrR proteins might lead to
cell death and failure of cell division, while pSC101 has a very low copy number, allowing fewer ChrR proteins to be
produced. pSC101 is ideal for YieF as well because it prevents overexpression of YieF proteins, allowing a stable gene
dosage at physiological levels across all cells (Thompson, 2018).

'
},{
"title": "2.2 Plasmid Construction for Degradation
Systems",
"content": '

Through the results of the Electrophoresis of genes,
we are able to determine whether we have successfully amplified copies of protein genes using PCR. The image of ChrR
displays two sharp, clean bands at 634 bp, meaning that the primers from PCR have successfully found and amplified the
exact region of ChrR; The image of YieF displays a sharp, clean band at 640 bp, meaning that the primers from PCR have
successfully found and amplified the exact region of YieF.

![D:/LYJ/2025-iGEM/李琦-构建可还原六价铬毒性的大肠杆菌工程菌株/最终实验结果汇总/PCR扩增、验证、测序、菌落图/ChrR-YieF 扩增图_画板 1.jpgChrR-YieF 扩增图_画板 1](https://static.igem.wiki/teams/5737/result/17.webp)

**Figure 16. Gel electrophoresis results of ChrR and YieF Genes**

According to Figure 17, there is a clean, sharp band
at 10498 bp, which is consistent with the base pair number in pSC101\'s results, meaning that the pSC101 plasmid has
been successfully cut into a linear form. The absence of any high molecular bands confirms that the digestion went to
completion without partial activity.

![pSC101 Linear 双酶切图](https://static.igem.wiki/teams/5737/result/18.webp)

**Figure 17. Results of gel electrophoresis from the double enzyme digestion of pSC101**

As shown in Figure 18A, the bacteria have a steady
growth rate, with pSC101-ChrR having lower densities, pSC101-YieF and pSC101-ChrR-YieF having relatively higher
densities. This highlights that pSC101-YieF and pSC101-ChrR-YieF have a higher success rate in plasmid assembly, while
pSC101-ChrR has a lower success rate in plasmid assembly. Yet, all four plates display signs of bacterial growth,
proving the success of inserting the plasmids with the corresponding E. coli, which is further testified in the bottom
series of images.

Based on Figure 18B, the image of pSC101-ChrR
displays three sharp, clean bands at 634 bp, meaning that the gene ChrR has successfully inderted into the pSC101
plasmid; the image of pSC101-ChrR displays five sharp, clean bands at 640 bp, meaning that the gene YieF has
successfully inderted into the pSC101 plasmid; the image of pSC101-ChrR-YieF displays ten sharp, clean bands at 2640
bs, meaning that the combination pair f ChrR-YieF has successfully inderted into the pSC101 plasmid.

![降解菌 菌落鉴定组图_画板 1](https://static.igem.wiki/teams/5737/result/19.webp)

**Figure 18. Results of Recombinant Plasmid Identification**

The extracted, recombined plasmid, along with the F1
and R1 primers, was then sent to a genomic sequence testing company, Tsingke. The results we received are displayed as
such:

![测序组图_画板 1](https://static.igem.wiki/teams/5737/result/20.webp)

**Figure 19. The DNA sequencing results**

After gene sequencing of the desired gene, it has
once again been verified, matching the results from the colony PCR gel electrophoresis as well as the initial gel
electrophoresis that confirms gene PCR amplification. Thus, through these two instances of verification, we
successfully proved that our recombined plasmid contains our desired gene, and that said recombined plasmid exists in
multiple *E. coli* cell colonies. As a result, the cloning of the pSC101-ChrR\pSC101-YieF\pSC101-ChrR-YieF
plasmid was successful.

'
},{
"title": "2.3 Protein Expression and Purification",
"content": '

**2.3.1 Recombinant plasmid transformation into
*E. coli* BL21(DE3)**

We inserted our 3 target genes(pSC101-ChrR,
pSC101-YieF, and pSC101-ChrR-YieF)containing araBAD promoters into the *E. coli* BL21(DE3) in order to produce
moderate amounts of protein for protein-related experiments. BL21(DE3) is a form of B strain *E. coli* with a
mutation on the lon and ompT genes. This results in a lack of the lon and ompT proteases within the *E. coli,*
which allows for increased protein expression (Jeong, Kim, Li, 2015). BL21(DE3) hence provides moderate expression of
genes with strong *E. coli* promoters, such as the araBAD promoter - the one on our recombinant plasmids.

The three culture plates
below indicate the growth of *E. coli* pSC101-ChrR-BL21, pSC101-YieF-BL21, and pSC101-ChrR-YieF-BL21 incubated
at 37°C for 12 hours.

![菌落组合图_画板 1](https://static.igem.wiki/teams/5737/result/21.webp)

**Figure 20. The culture plates mentioned above (from left to right: pSC101-ChrR-BL21, pSC101-YieF-BL21,
pSC101-ChrR-YieF BL21)**

The results from PCR and gel-electrophoresis below
indicate that the target genes (ChrR, YieF, and ChrR-YieF) are present in the DNA of *E. coli* BL21(DE3). There
is DNA present that matches the size of these genes.

![pcr组图_画板 1](https://static.igem.wiki/teams/5737/result/22.webp)

**Figure 21. The PCR results mentioned above (from left to right: pSC101-ChrR, pSC101-YieF,
pSC101-ChrR-YieF)**

**2.3.2** **Protein Expression,
Purification, and Characterization**

These SDS-PAGE results indicate how proteins from our
3 target genes (ChrR, YieF, and ChrR+YieF protein) were successfully expressed in the *E. coli* BL21(DE3). There
are proteins present that are the same size as our target proteins.

![蛋白图_画板 1](https://static.igem.wiki/teams/5737/result/23.webp)

**Figure 22. The SDS-PAGE results mentioned above (from left to right: ChrR, YieF, ChrR+YieF
)**

'
},{
"title": "2.4 Chromium Resistance",
"content": '

Before testing out specific data regarding how well
each target protein (ChrR, YieF, and ChrR+YieF protein) can detect chromium (via visual signals) and degrade chromium,
we first checked our *E. coli*\'s overall resistance to different concentrations of chromium over time.

To test how resistant bacteria were to Cr VI, we
measured the amount of bacteria in units of optical density 600. Optical density 600 (OD600) is a measure of light
scattering caused by the presence of particles in a solution or culture media. Specifically, in our experiment, we
measured the light scattering of E. coli. The more E. coli are in the solution, the higher the value of OD600 would be
(Diaz, Viplav 2023).

The figure displays OD600 measurements for three
engineered bacterial strains at varying hexavalent chromium concentrations. Data analysis indicates that within the
0–80 mg/L concentration range, both pSC101-ChrR and pSC101-YieF strains exhibit robust growth capacity, maintaining
stable growth rates and accumulating significant biomass. However, when concentrations reached 100 mg/L or higher,
bacterial growth was markedly inhibited, with growth rates significantly declining and final biomass levels
stabilizing. This indicates that concentrations at or above this threshold approached or reached the upper limit of
growth.

However, the maximum tolerance of the
pSC101-ChrR-YieF strain is 50 mg/L. When Cr(VI) concentrations reach 80 mg/L or higher, the strain ceases to grow.

![组图_画板 1](https://static.igem.wiki/teams/5737/result/24.webp)

**Figure 23. The change in OD over 6h for pSC101-chrR, pSC101-YieF, pSC101-chrR-YieF in different Cr(VI)
solution**

Notably, we summarized the above data at 6 hours (the
maximum time) into one bar graph, and based on the two data sets, we noted how:

1) Up until 50mg/L of Cr(VI), the optical density for
all 3 *E. coli* groups was generally increasing. After 50mg/L of Cr(VI), the optical density for all 3
*E. coli* groups was generally decreasing.

2) By looking at bacterial growth after 6h in Cr(VI)
concentrations, the YieF protein had relatively higher optical density than the ChrR and ChrR-YieF protein for 0-50
mg/L. After 50mg/L, optical densities of all 3 proteins significantly decreased, and the highest optical density
protein varied.

![](https://static.igem.wiki/teams/5737/result/25.webp)

**Figure 24. The bacterial growth in different Cr(VI) solutions after 6 hours**

'
},{
"title": "2.5 Chromium Degradation",
"content": '

**2.5.1 Chromium Degradation**

This series of experiments was aimed at testing how
well each target protein (ChrR, YieF, and the ChrR+YieF protein) can degrade chromium. We analyzed concentration
changes before and after degradation of Cr(VI) to calculate a reduction rate for each degrading protein.

This series of experiments was out-of-cell (in
vitro), using solutions of each target protein (ChrR, YieF, and ChrR+YieF) with Cr(VI) at different concentrations (0,
10, 30, 50, 80, 100 mg/L Chromium VI).

We isolated our 3 target proteins (ChrR, YieF, and
ChrR+YieF protein) and added the protein solution under different Cr(VI) levels for different periods of time, and
transferred the solutions to read at A540 nm absorbance.

To determine chromium concentrations, we read the
absorbance values of the different solutions.

***A =* Ɛ*cl***

**Note: A: absorbance value; Ɛ: extinction
coefficient; C: concentration of samples; L: path length of micropipette plate**

Based on the above formula, we determined the
specific absorbance values. Absorbance is directly proportional to concentration, so the higher the absorbance value,
the higher the concentration of chromium in the protein solution. The graphs below indicate our results.

![组图_画板 1](https://static.igem.wiki/teams/5737/result/26.webp)

**Figure 25. Cr(VI) concentrations in ChrR, YieF, and ChrR+YieF protein solutions after degradation**

The above graphs all share the same trends: For every
target protein, as time passed, the chromium concentration for every initial concentration group (0-100) decreased
continuously.

Through this, we came to our second conclusion: the
concentrations all decreased the most from the 0 to 5 hr mark, thereby **making 5 hours the optimum time for
degrading chromium**.

### **2.5.2 Reduction Rate of each Protein**

We then calculated the rate of reduction (degradation
rate) for every target protein (ChrR, YieF, and ChrR+YieF) for every concentration. Based on our previous result of 5
hours being the optimum time, we measured degradation at the 5-hour mark. The graph below shows the reduction
(degradation) rates for ChrR, YieF, and ChrR+YieF.

After 5 hours of reaction, YieF protein demonstrated
significantly superior degradation efficacy compared to ChrR and ChrR+YieF, achieving a higher degradation rate. This
indicates that YieF is the most potent chromium-degrading protein with substantial application value.

![3个蛋白比较 combined bar](https://static.igem.wiki/teams/5737/result/27.webp)

**Figure 26.Graph for the degradation of Cr(VI) at different concentrations after 5 hours**

Subsequently, we further calculated the actual
efficiency of the three proteins in reducing chromium. YieF’s reduction rate reached 78.6%, which was higher than
ChrR, which was higher than ChrR+YieF. Paired with our previous conclusion, we came to our third conclusion: YieF
exhibits the highest rate of Cr(VI) degradation at 12 hours, so it is the most optimal protein for hexavalent chromium
degradation.

**Table 2: Calculation of Reduction Rates for
Three Types of Protein Chromium**

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| **Cr(VI)umol/mL** | **0h** | **5h** | **12h** | **0-5h** | **0-12h** |
| **ChrR** | 0.016906475 | 0.00647482 | 0.5395683 | 69.41% | 74.5% |
| 0.033093525 | 0.00881295 | 0.008273381 |
| 0.04028777 | 0.014928058 | 0.009172662 |
| 0.035791367 | 0.010611511 | 0.010071942 |
| 0.053956835 | 0.011510791 | 0.010432 |
| **YieF** | 0.017625899 | 0.006294964 | 0.557554 | **72.5%** | **78.6%** |
| 0.028057554 | 0.01205036 | 0.8992806 |
| 0.030035971 | 0.007194245 | 0.002697842 |
| 0.054856115 | 0.010071942 | 0.010071942 |
| 0.066366906 | 0.010971223 | 0.010611511 |
| **ChrR+YieF** | 0.017985612 | 0.007553957 | 0.00557554 | 54.6% | 69.3% |
| 0.029136691 | 0.009352518 | 0.007374101 |
| 0.031834532 | 0.014208633 | 0.00881295 |
| 0.033093525 | 0.020683453 | 0.012230216 |
| 0.039568345 | 0.018165468 | 0.01294964 |

'
}]
}, {
"title": "Future Discussion",
"content": '

Our results indicate that the best time was 5 hours,
the highest concentration at which our proteins performed optimally was 50mg/L, and the best gene and protein for
doing so was YieF. This is sufficiently above various national standards for chromium concentrations in industrial
wastewater: China: 0.5 mg/L, USA: 0.1 mg/L, Europe: 0.05 mg/L.

In the chromium detection experiment, we tested the
blueness of our chromium-containing bacterial solutions by visualizing the degree of dark blueness. Though we
performed various experiments afterwards to verify that the tube with the darkest blue visual appearance is indeed the
one with the most optimal protein performance, we could develop a formula that directly links the blueness (caused by
amilCp proteins) to protein performance potential.

We chose *E. coli* for our experiment because
it is: Cheap (good for lowering prices of our hardware product), Easy to engineer or do biosynthetic experiments on,
Protists can easily create recombinant plasmids, therefore it can easily produce our target proteins, *E. coli*is relatively survivable in toxic Cr(VI) environments. In addition, there is a potential for us to use a similar
experiment logic to degrade other heavy metals. Rhamnose can reduce metal ions itself at a specific acidic pH, and can
promote reduction in proteins, not only for chromium reductases.

In summary, we have successfully developed an
integrated biological approach capable of detecting and efficiently degrading heavy metal Cr(VI) from scratch. Its
functional feasibility and treatment efficacy have been fully validated in experimental systems. This system
integrates a synthetic biology-based chrB-amilCP biosensor with a highly efficient soluble flavoprotein reduction
module, enabling specific visual detection and environmentally friendly reduction conversion of Cr(VI). This research
provides a novel technical pathway and experimental basis for in situ monitoring and green remediation of heavy
metal-contaminated water bodies, demonstrating potential application prospects and dissemination value in the field of
environmental biotechnology.

'
},{
"title": "References",
"content": '

Ackerley, D. F., et al. “Chromate-Reducing Properties
of Soluble Flavoproteins from Pseudomonas Putida and Escherichia Coli.” *Applied and Environmental
Microbiology*, vol. 70, no. 2, Feb. 2004, pp. 873-882, https://doi.org/10.1128/aem.70.2.873-882.2004. Accessed
17 Nov. 2020.

Baldiris, Rosa, et al. “Reduction of Hexavalent
Chromium and Detection of Chromate Reductase (ChrR) in Stenotrophomonas Maltophilia.” *Molecules*, vol. 23, no.
2, 13 Feb. 2018, p. 406, https://doi.org/10.3390/molecules23020406.

Das, Manimita, et al. *Microbial Chromium
Degradation: Biological Evolution, Mitigation, and Mechanism*. 2015.

Diaz, Sara, and Abhiyan Viplav. “Unlocking OD600
Assay and Bacterial Culture Growth: 4 Facts and 4 Tips | Byonoy Journal.” *Compact Microplate Reader | Absorbance
96*, 5 Nov. 2023, byonoy.com/journal/od600-4facts-4tips-bacterial-culture-growth/.

Jeong, Haeyoung, et al. “Complete Genome Sequence of
Escherichia Coli Strain BL21.” *Genome Announcements*, vol. 3, no. 2, 19 Mar. 2015,
www.ncbi.nlm.nih.gov/pmc/articles/PMC4395058/, https://doi.org/10.1128/genomea.00134-15.

Sala, Luis F, et al. “Oxidation of L-Rhamnose and
D-Mannose by Cr(VI) in Perchloric Acid. A Comparative Study.” *Canadian Journal of Chemistry*, vol. 70, no. 7,
1 July 1992, pp. 2046–2052, https://doi.org/10.1139/v92-258. Accessed 22 June 2025.

'
}]%}
{% block title %}Results{% endblock %}
{% block lead %}

![](https://static.igem.wiki/teams/5737/banner/v1/wet-lab.webp)
![](https://static.igem.wiki/teams/5737/common/wetlab-1.webp)

WET LAB

RESULTS

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{% block name %}wetlab{% endblock %}