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.

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.

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(CrO₄^2-, Cr₂O₇^4-), 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.

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.

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.

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.

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).

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.

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.

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:

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.

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:

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.

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:

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).

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.

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.

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.

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).

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.

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.

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.

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:

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.

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.

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.

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.

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

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.

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.

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

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 = Ɛcl

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.

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.

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