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 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.

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(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 3. Plasmid Map of pET28a-chr-T7-amilCp

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 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.



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).



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:



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.




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:



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.

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 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).

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.

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.

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.

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.

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 prevents the 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.

Figure 13. Plasmid Map of pSC101-chrR

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.

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.

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.

Figure 16. pSC101-ChrR Colony Monoclonal Identification Chart

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:



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).



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.



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.



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.



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.



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.



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

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.

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.

Figure 24. Plasmid Map of pSC101-YieF

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.

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.

Figure 26. pSC101-YieF Colony Monoclonal Identification Chart

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.

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 pSC101-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.

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.

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.

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.

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

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.

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.

Figure 32. Plasmid Map of pSC101-ChrR-YieF

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.

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.

Figure 34. pSC101-ChrR Colony Monoclonal Identification Chart

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.

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.

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.

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.

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.

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

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.