Name: pSC101

Base Pair: ~10498 bp

Origin: E. coli DH5α

Properties: The first cloning vector ever used. It is a low-copy plasmid vector carrying a selectable marker that allows the stable maintenance of inserted genes.

Usage and biology:

pSC101 is a stable plasmid backbone that has a low genome copy number (4~6 copies/chromosome)[1], which reduces the metabolic stress on the host and prevents overexpression toxicity.

Figure 1. Plasmid Map of pSC101

Moreover, pSC101 lacks the transfer origin, oriT, so genes in the bacteria cannot spread via conjugation with other bacteria, increasing biosafety. pSC101 is very inefficient at replicating in temperatures above 37 °C. This means that the plasmid is rapidly lost from the host cell when temperature shifts, making sure that the genetic material it carries cannot persist outside of a controlled lab environment. The kanR gene provides kanamycin resistance, which is very helpful to extract only your intended bacteria from a kanamycin petri dish, killing off all other unwanted bacteria. Most importantly to our project, the rhamnose-induced pathway could precisely control gene expression.

The rhaSR operon codes for RhaS regulator protein(positive transcriptional activator, switches operon on when L-rhamnose is present) and RhaR protein(activates transcription of PrhaSR promoter in the presence of rhamnose) with a promoter PrhaSR at the start.

Figure 2. Diagram of PrhaSR Operon and rhaB Operon with l-rhamnose Not Present

The RhaR gene is always expressed at a low but consistent level, which produces low amounts of RhaR protein. Only when L-rhamnose is present does it bind to RhaR and activate RhaR. The activated RhaR protein then binds upstream of the PrhaSR promoter, which turns on transcription of the rhaSR operon. [3]

Figure 3. Diagram of PrhaSR Operon and rhaB Operon with l-rhamnose Not Present

The rhaS gene inside the operon produces RhaS protein, which becomes active when bound by L-rhamnose. Active RhaS then binds upstream of the rhaB promoter and helps RNA polymerase efficiently initiate transcription of downstream genes.

References:

[1] Hashimoto-Gotoh T, Franklin FC, Nordheim A, Timmis KN. Specific-purpose plasmid cloning vectors. I. Low copy number, temperature-sensitive, mobilization-defective pSC101-derived containment vectors. Gene. 1981 Dec;16(1-3):227-35. doi: 10.1016/0378-1119(81)90079-2. PMID: 6282694.

[2] Tobin, J. F., & Schleif, R. F. (1987). Positive regulation of the Escherichia coli L-rhamnose operon is mediated by the products of tandemly repeated regulatory genes. Journal of Molecular Biology, 196(4), 789–799.

[3] Egan, S. M., & Schleif, R. F. (1993). A regulatory cascade in the induction of rhaBAD. Journal of Molecular Biology, 234(1), 87–98.

Name: pchr pro-chrB

Base Pair: ~1108 bp

Origin: O. tritici (5bvl1)

Properties: The chromate-sensing regulator chrB is a genetic part that encodes the ChrB repressor protein and controls transcription of downstream genes.

Usage and biology:

chrB gene encodes a chromate sensing transcriptional regulatory protein from O.tritici(5bvl1) which is known for its strong resistance against Chromate(CrO₄^2-)[1]. The chrB gene's most important function is being a bacterial biosensor for Cr(VI): a heavy metal that is highly carcinogenic and toxic.

The chrB gene's core function is to produce a Cr(VI)-responsive repressor protein.

Figure 4. chrB Gene Without Cr(VI) Present, Focusing on: p_chr Operator, chrB Gene, and T7 Promoter in the Regulatory Module chr

The chrB gene is consistently expressed and continuously produces repressor protein ChrB. 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. 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.

Figure 5. chrB Gene with Cr(VI) Present, Focusing On: p_chr Operator, chrB Gene, and T7 Promoter in the Regulatory Module chr

The primary application of the gene is used in synthetic biology as a monitor and reporter of environmental circumstances. By placing a reporter gene (e.g., amilCP for visible color detection) downstream of the ChrB-regulated p_chr operator, the bacteria act as a sensor that provides a simple, inexpensive, and visual response when Cr(VI) contaminates water and soil samples.

References:

[1] Branco, R., & Morais, P. V. (2013). Identification and characterization of the transcriptional regulator ChrB in the chromate resistance determinant of Ochrobactrum tritici 5bvl1. PLOS ONE, 8(11), e77987.

Name: ChrR

Base Pair: ~634 bp

Origin: Pseudomonas putida (P. putida) KT2440

Properties: a flavoprotein-chromate-reductase that converts Cr(VI) into Cr(III).

Usage and biology:

ChrR has a large pH and temperature-tolerant range. ChrR is also a dimer that possesses chromate reductase function, indicating that it can effectively reduce chromate salts to Cr(III). During reduction, ChrR will produce flavin semiquinone intermediate, resulting in a situation where more than 25% of the NADH electrons transform into ROS—reactive oxygen species (which can cause damage to "proteins, lipids, and nucleic acids, leading to cell and tissue injury"). However, in return, ChrR is able to allow P. putida to be exempted from the toxins of chromate salts.

Reduction mechanism:

Starting from Cr(VI), Chromate reductase can catalyze the reaction of transferring two electrons: one electron to Cr(VI) to form Cr(V), while the other electron generates ROS.

Figure 6. Diagram Display of ChrR Degrading Cr(VI) to Cr(V)

The second electron transfer fully reduces Cr(V) to Cr(III). One inefficient process about the reduction process is that a proportion of Cr(V) is able to regenerate ROS on its own, and how Cr(VI) and Cr(V) are able to constantly change their valence states due to the presence of "one electron shuttle". Such oxidative stress results in more energy wasted during the process of reducing Cr(VI) to Cr(III) [1].

Figure 7. Diagram Display of ChrR Degrading Cr(V) to Cr(III)

Cultivation:

Figure 8 represents the gel electrophoresis diagram for the ChrR gene. The brightness of the band around 634 bp indicates a relatively high concentration of YieF detected near 634 bp.

Figure 8. Gel Electrophoresis Diagram for ChrR Gene

References:

[1] Baldiris, R., Acosta-Tapia, N., Montes, A., Hernández, J., & Vivas-Reyes, R. (2018). Reduction of Hexavalent Chromium and Detection of Chromate Reductase (ChrR) in Stenotrophomonas maltophilia. Molecules (Basel, Switzerland), 23(2), 406.

Name: YieF

Base Pair: ~640 bp

Origin: E. coli MG1655

Properties: YieF, similar to ChrR, is also capable of reducing chromate salts to Cr(III). However, unlike ChrR, the reduction process does not result in the production of flavin semiquinone intermediate, and only 25% of the NADH electrons are used to create ROS.

Reduction mechanism:

YieF can directly transfer four electrons to Cr(VI), forming Cr(III) directly. This meant that there were no Chromate ions left in the unstable state of Cr(V), and moving back and forth between Cr(V) and Cr(VI) valence states. After the three electrons are successfully transferred to Cr(VI) to form Cr(III), the leftover electron forms ROS with oxygen in the surroundings.

Figure 9. Diagram Display of YieF Degrading Cr(VI) to Cr(III)

Cultivation:

Figure 10 represents the gel electrophoresis diagram for the YieF gene. The brightness of the band around 640 bp indicates a relatively high concentration of YieF detected near 640 bp.

Figure 10. Gel Electrophoresis Diagram for YieF Gene

References:

[1] Baldiris, R., Acosta-Tapia, N., Montes, A., Hernández, J., & Vivas-Reyes, R. (2018). Reduction of Hexavalent Chromium and Detection of Chromate Reductase (ChrR) in Stenotrophomonas maltophilia. Molecules (Basel, Switzerland), 23(2), 406.

Name: pET28a-chr-T7-amilCP

Base Pair: ~6992 bp

Construction Design:

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. [1]

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

Figure 11. 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 12. 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.[2]

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 13. Plasmid Map of pET28a-chr-T7-amilCp

Engineering Principle:

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.

1. 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 14. chr-T7-amilCP Pathway Under No Cr(VI) Presence

2. 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.

3. Cr(VI) inducing 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 15. chr-T7-amilCP Pathway with Cr(VI) Present

4. 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.

Experimental Approach, Cultivation:

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 16. Gel Electrophoresis Diagram for Linearized pET28a

Figure 16 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 17. Gel Electrophoresis Diagram for Gene chr-T7-amilCP

In Figure 17, 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 [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. [6]

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 18. 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 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 19. 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 20. 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.

Characterization, Measurement:

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 21. 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 22. 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 23. 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 24. 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.

References:

[1] Novagen. PET System Manual,

research.fredhutch.org/content/dam/stripe/hahn/methods/biochem/pet.pdf.

[2] Rosano, G. L., & Ceccarelli, E. A. (2014). Recombinant protein expression in Escherichia coli: advances and challenges. Frontiers in Microbiology, 5, 172.

[3] Cervantes, C., Campos-García, J., Devars, S., Gutiérrez-Corona, F., Loza-Tavera, H., Torres-Guzmán, J. C., & Moreno-Sánchez, R. (2001). Interactions of chromium with microorganisms and plants. FEMS Microbiology Reviews, 25(3), 335–347.

[4] Pepi, M., & Baldi, F. (1992). Modulation of chromium(VI) toxicity by organic and inorganic sulfur species in yeasts from industrial wastes. Biometals, 5(3), 179–185.

[5] Kostylev, Maxim et al. “Cloning Should Be Simple: Escherichia coli DH5α-Mediated Assembly of Multiple DNA Fragments with Short End Homologies.” PloS one vol. 10,9 e0137466. 8 Sep. 2015, doi:10.1371/journal.pone.0137466.

[6] Thermo Fisher Scientific. (n.d.). DH5α competent cells. Retrieved October 26, 2023.

Name: pSC101-ChrR

Base Pair: ~11132bp

Construction Design:

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 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 25. Plasmid Map of pSC101-chrR

Engineering Principle:

Our purpose for pSC101-ChrR is to create a flavoprotein that can remove Cr(VI) in water. Since pSC101 is a Rhamnose-induced pathway, the downstream genes will only be activated once the bacteria are able to detect Rhamnose in the surroundings. Through this mechanism, we allow pSC101-ChrR to be expressed under conditions with Rhamnose present, activating the ChrR gene so that the bacteria will be able to degrade Cr(VI) to Cr(III).

Experimental Approach, Cultivation:

1. Double-digested pSC101 plasmid backbone

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.



Figure 26. Gel Electrophoresis Diagram for Linearized pSC101

The gel electrophoresis diagram above indicates that the linearized pSC101 plasmids are roughly around 10498 bp, as the glowing band is higher in place than the 8000 bp marker. From the diagram above, the intensity and brightness of the pSC101's band are relatively low, in comparison with the marker. However, its concentration remains sufficient for subsequent experiments to proceed normally.

2. ChrR PCR Amplification

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.



Figure 27. Gel Electrophoresis Diagram for Gene ChrR

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.

3. Homologous Recombination and Heat Shock Transfer

We then set up a reaction mixture for inserting our desired gene into the linear pSC101 vector. Similarly, we realized that adding a small amount of E. coli competent cells, along with the plasmid and desired gene, can serve as an in vivo homologous recombination method. However, pSC101 is a low-copy-number plasmid that is very sensitive to the host's genetic background[1], so we chose to use EPI400. EPI400 is a recA strain, where its RecA protein can stop plasmid rearrangement or loss, just as Thermo Fisher Scientific(invitrogen) Strain Documentation said "EPI400 is designed for the stable propagation of low-copy-number plasmids".

We chose to add kanamycin into 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 28. Cell Culture Display of EPI400 Bacteria with pSC101-ChrR 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.

4. Colony PCR Identification

5 colonies from each agar plate were chosen, and we used our pipette to dab some of the colony to place it in a PCR mix, along with primers (F1, forward; and R1, reverse) that define the amplification region. The mixture is then placed into a PCR machine, and the final products are loaded into an agarose gel for gel electrophoresis. Previous gel electrophoresis used to confirm successful ChrR amplification verifies that the gene should be around 634 bp. The glowing band results indicated below show roughly 634 bp as well, indicating successful insertion of the gene into pSC101, and the successful formation of pSC101-ChrR.



Figure 29. Gel Electrophoresis Diagram from the Colony PCR of Bacteria in an Agar Plate

5. Gene Sequencing

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 30. Gene Sequencing of pSC101-ChrR

After gene sequencing of the desired gene, it has once again been verified to be around 634 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.

Characterization, Measurement:

1. Heat Shock Transformation of pSC101-ChrR 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 these two proteins' presence allows for increased 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 31. 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).



Figure 32. Gel Electrophoresis Diagram from the Colony PCR of Bacteria in an Agar Plate

The results show that it is around 634bp, matching the prior PCR bp length, confirming that we successfully transferred pSC101-ChrR into BL21(DE3)

2. Resistance to chromium

After cultivating the competent plasmid-containing cells, the cells are used to conduct the bacteria-chromium-resistance-test for pSC101-ChrR bacterium while adding antibiotics to kill off other bacteria. We study bacteria growth from 0-6hrs under concentrations of 0mg/L, 10mg/L, 30mg/L, 50mg/L, 80mg/L, and 100mg/L of chromium at 37 °C, 220 rpm. We take samples of the bacteria-chromium solution at the end of each hour(starting from 0) and put them into smaller tubes to do an OD600 measurement. The higher the OD600, the more bacteria are thriving in the solution. The tubes that served as the bacteria's culture medium were photographed and also spread on a petri dish for more vivid qualitative analysis. 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 33. 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 34. 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 35. 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. Protein Extraction

To get the protein out, we did a series of steps to disrupt the cell and complete cell lysis using a centrifuge, a lysosome, and a Sonicator. We pipette 2mL of the finished sample into 2mL tubes, obtaining the crude protein. The rest of the sample is pipetted into 15 mL tubes for purification. Add 1 mL Ni-NTA into the tubes. Having both crude and pure protein, we prepare to run an SDS-PAGE.



Figure 36. SDS-PAGE Diagram of ChrR Crude (Left Region) and Pure (Right Region)

After checking, we found that the ChrR proteins are around 24 kDa, which means they match the protein's specific molecular weight, implying the successful production of ChrR.

4. Quantification

The degradation of Cr(VI) by ChrR was quantified by measuring residual Cr(VI) concentrations with a colorimetric assay. In this assay, 1,5-diphenylcarbazide reacts with Cr(VI) in an acidic solution to form a purplish-red complex that absorbs light at 540 nm. The addition of the ChrR solution that is mixed with Cr(VI) with reagents 1 and 2 results in an acidic solution with a purple-red color. The higher the rate of degradation of Cr(VI), the lighter the color of the tube is, since the concentration of Cr(VI) decreases as the production of Cr(III) forms(equilibrium).



Figure 37. Combined Graph of ChrR Protein's Cr(VI) Degradation Under Different Cr(VI) Concentrations

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.

References:

[1] Ackerley, D. F., Gonzalez, C. F., Park, C. H., Blake, R., Keyhan, M., & Matin, A. (2004). Chromate-reducing properties of soluble flavoproteins from Pseudomonas putida and Escherichia coli. Applied and Environmental Microbiology, 70(2), 873–882.

Name: pSC101-YieF

Base Pair: ~11138bp

Construction Design:

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 38. Plasmid Map of pSC101-YieF

Engineering Principle:

pSC101-YieF is a Rhamnose-inducing pathway, indicating that the YieF gene is expressed by the bacteria once the bacteria detect the presence of Rhamnose in the surrounding. Thus, the addition of Rhamnose in the liquid that includes Cr(VI) activates the YieF gene, engendering the process of degrading Cr(VI).

Experimental Approach, Cultivation:

1. Double-digested pSC101 plasmid backbone

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.



Figure 39. Gel Electrophoresis Diagram for Linearized pSC101

The gel electrophoresis diagram above indicates that the linearized pSC101 plasmids are roughly around 10498 bp, as the glowing band is higher in place than the 8000 bp marker. From the diagram above, the intensity and brightness of the pSC101's band are relatively low, in comparison with the marker. However, its concentration remains sufficient for subsequent experiments to proceed normally.

2. YieF PCR Amplification

Our desired gene in this experiment was YieF, which was initially amplified by Polymerase Chain Reaction (PCR) amplification to produce many copies of the 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 40. Gel Electrophoresis Diagram for Gene YieF

Similarly, the glowing bands indicate that the YieF DNA has been successfully amplified, as its band is visualized around ~640 bp, indicating that it follows the expected size of the gene. Thus, these successfully amplified genes will be used in homologous recombination.

3. Homologous Recombination and Heat Shock Transfer

We then set up a reaction mixture for inserting our desired gene into the linear pSC101 vector. Similarly, we realized that adding a small amount of E. coli competent cells, along with the plasmid and desired gene, can serve as an in vivo homologous recombination method. However, pSC101 is a low-copy-number plasmid that is very sensitive to the host's genetic background[1], so we chose to use EPI400. EPI400 is a recA strain, where its RecA protein can stop plasmid rearrangement or loss, just as Thermo Fisher Scientific(Invitrogen) Strain Documentation said, "EPI400 is designed for the stable propagation of low-copy-number plasmids".

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 41. Cell Culture Display of EPI400 Bacteria with pSC101-YieF 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.

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, adding 8ul of ddH₂O and 10ul of 2x-mix to all the tubes along with primers (YieF-F2, forward; and YieF-R2, reverse) that define the amplification region. The previous gel electrophoresis used to confirm successful YieF amplification verifies that the gene should be around ~640 bp. The glowing band results indicated below show roughly 640 bp, indicating successful insertion of the gene into pSC101, and the successful formation of pSC101-YieF.




Figure 42. Gel Electrophoresis Diagram from the Colony PCR of Bacteria in an Agar Plate

5. Gene Sequencing

The extracted, recombined plasmid, along with the F2 and R2 primers, was then sent to a genomic sequence testing company, Tsingke. The results we received are displayed as such:



Figure 43. Gene Sequencing of pSC101-YieF

After genomic sequencing of the desired gene, it has once again been verified to be around 640 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.

Characterization/Measurement:

1. Heat Shock Transformation of pSC101-YieF 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 these two proteins' presence allows for increased protein expression. We successfully transformed the pSC101-YieF into E. coli BL21(DE3) using the heat shock transformation method. After roughly 15 hours, the resulting cell culture was displayed as such:



Figure 44. Cell Culture Display of BL21(DE3) Bacteria with pSC101-YieF Gene

After inoculation and incubation of the bacteria, we did another PCR test to check if the plasmid was successfully transferred into the E. coli BL21(DE3).



Figure 45. Gel Electrophoresis Diagram from the Colony PCR of Bacteria in an Agar Plate

The results show that it is around 640 bp, matching the prior PCR bp length, confirming that we successfully transferred pSC101-YieF into E. coli BL21(DE3).

2. Resistance to chromium

After cultivating the competent plasmid-containing cells, the cells are used to conduct the bacteria-chromium-resistance-test for the pSC101-YieF bacterium while adding antibiotics to kill off other bacteria. We study bacteria growth from 0-6hrs under concentrations of 0mg/L, 10mg/L, 30mg/L, 50mg/L, 80mg/L, and 100mg/L of chromium at 37 °C, 220 rpm. We take samples of the bacteria-chromium solution at the end of each hour(starting from 0) and put them into smaller tubes to do an OD600 measurement. The higher the OD600, the more bacteria are thriving in the solution.

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 46. 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 47. 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 48. 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

3. Protein Extraction

To get the protein out, we did a series of steps to disrupt the cell and complete cell lysis using a centrifuge, a lysosome, and a Sonicator. We pipette 2 mL of the finished sample into 2 mL tubes, obtaining the crude protein. The rest of the sample is pipetted into 15 mL tubes for purification. Add 1 mL Ni-NTA into the tubes. Having both crude and pure protein, we prepare to run an SDS-PAGE.



Figure 49. SDS-PAGE Diagram of YieF Crude (Left Region) and Pure (Right Region)

After checking, we found that the YieF proteins are around 24 kDa, which means they match the protein's specific molecular weight, implying the successful production of YieF.

4. Quantification

The degradation of Cr(VI) by YieF was quantified by measuring residual Cr(VI) concentrations with a colorimetric assay. In this assay, 1,5-diphenylcarbazide reacts with Cr(VI) in an acidic solution to form a purplish-red complex that absorbs light at 540 nm. The addition of the YieF solution that is mixed with Cr(VI) with reagents 1 and 2 results in an acidic solution with a purple-red color. The higher the rate of degradation of Cr(VI), the lighter the color of the tube is, since the concentration of Cr(VI) decreases as the production of Cr(III) forms(equilibrium).



Figure 50. Combined Graph of YieF Protein's Cr(VI) Degradation Under Different Cr(VI) Concentrations

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.

References:

[1] Cabello, F., Timmis, K., & Cohen, S. N. (1976). Replication control in a composite plasmid constructed by in vitro linkage of two distinct replicons. Nature, 259(5541), 285-290.

Name: pSC101-ChrR-YieF

Base Pair: ~11841bp

Construction Design:

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 51. Plasmid Map of pSC101-ChrR-YieF

Engineering Principle:

Since the backbone of this bacterium is derived from pSC101, this indicates that it is a Rhamnose-inducing pathway. Moreover, there are two Cr(VI)-degrading genes located downstream, which can be activated by the activation of the rhaB promoter. Under the condition that Rhamnose is able to be detected by the bacteria from the surroundings, the rhaB promoter is initiated, leading to the expression of ChrR and YieF genes. This results in the production of ChrR and YieF proteins, degrading Cr(VI) present in the surroundings.

Experimental Approach, Cultivation:

1. Double-digested pSC101 plasmid backbone

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.



Figure 52. Gel Electrophoresis Diagram for Linearized pSC101

The gel electrophoresis diagram above indicates that the linearized psis are roughly around 10498 bp, as the glowing band is higher in place than the 8000 bp marker. From the diagram above, the intensity and brightness of the pSC101's band are relatively low, in comparison with the marker. However, its concentration remains sufficient for subsequent experiments to proceed normally.

2. ChrR and YieF PCR Amplification

Our desired gene in this experiment was ChrR and YieF, which were initially amplified by Polymerase Chain Reaction (PCR) to produce many copies of the genes. 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.



Figure 53. Gel Electrophoresis Diagram for Gene ChrR

Similarly, the glowing bands indicate that the YieF DNA has been successfully amplified, as its band is visualized around ~640 bp, indicating that it follows the expected size of the gene. Thus, these successfully amplified genes will be used in homologous recombination.



Figure 54. Gel Electrophoresis Diagram for Gene YieF

3. Homologous Recombination and Heat Shock Transfer

We then set up a reaction mixture for inserting our desired gene into the linear pSC101 vector. Similarly, we realized that adding a small amount of E. coli competent cells, along with the plasmid and desired gene, can serve as an in vivo homologous recombination method. However, pSC101 is a low-copy-number plasmid that is very sensitive to the host's genetic background[1], so we chose to use EPI400.

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 55. Cell Culture Display of EPI400 Bacteria with pSC101-ChrR-YieF 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.

4. Colony PCR Identification

The gel electrophoresis used to confirm successful ChrR and YieF amplification verifies that the gene should be around ~1274 bp. The glowing band results indicated below show roughly 2640 bp. This is because between the ChrR and the YieF gene are many other genes, and the primers F2 and R2 are also added into the bp length, indicating successful insertion of the two genes into pSC101, and the successful formation of pSC101-ChrR-YieF.




Figure 56. Gel Electrophoresis Diagram from the Colony PCR of Bacteria in an Agar Plate

5. Gene Sequencing

The extracted, recombined plasmid, along with the F2 and R2 primers, was then sent to a genomic sequence testing company, Tsingke. The results we received are displayed as such:



Figure 57. Gene Sequencing of pSC101-ChrR-YieF

After gene sequencing the desired plasmid, it has once again been verified to be around 11841 bp, matching the results from our designed plasmid map. 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.

Characterization, Measurement:

1. Heat Shock Transformation of pSC101-ChrR-YieF 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 pSC101-ChrR-YieF into E. coli BL21(DE3) using the heat shock transformation method. After roughly 15 hours, the resulting cell culture was displayed as such:



Figure 58. Cell Culture Display of BL21(DE3) Bacteria with pSC101-ChrR-YieF Gene

After inoculation and incubation of the bacteria, we did another PCR test to check if the plasmid was successfully transferred into the E. coli BL21(DE3). The results show that it is around 2640bp, matching the prior PCR bp length, confirming that we successfully transferred pSC101-ChrR-YieF into BL21(DE3).



Figure 59. Gel Electrophoresis Diagram from the Colony PCR of Bacteria in an Agar Plate

2. Resistance to chromium

After cultivating the competent plasmid-containing cells, the cells are used to conduct the bacteria-chromium-resistance-test for the pSC101-ChrR-YieF bacterium while adding antibiotics to kill off other bacteria. We study bacteria growth from 0-6hrs under concentrations of 0mg/L, 10mg/L, 30mg/L, 50mg/L, 80mg/L, and 100mg/L of chromium at 37 °C, 220 rpm. We take samples of the bacteria-chromium solution at the end of each hour(starting from 0) and put them into smaller tubes to do an OD600 measurement. The higher the OD600, the more bacteria are thriving in the solution.

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 60. 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 61. 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 62. 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. Protein Extraction

To get the protein out, we did a series of steps to disrupt the cell and complete cell lysis using a centrifuge, a lysosome, and a Sonicator. We pipette 2 mL of the finished sample into 2 mL tubes, obtaining the crude protein. The rest of the sample is pipetted into 15 mL tubes for purification. Add 1 mL Ni-NTA into the tubes. Having both crude and pure protein, we prepare to run an SDS-PAGE.



Figure 63. SDS-PAGE Diagram of ChrR+YieF Crude (Left Region) and Pure (Right Region)

After checking, we found that the ChrR+YieF proteins are around 24 kDa, which means they match the protein's specific molecular weight, implying the successful production of ChrR+YieF.

4. Quantification

The degradation of Cr(VI) by ChrR+YieF was quantified by measuring residual Cr(VI) concentrations with a colorimetric assay. In this assay, 1,5-diphenylcarbazide reacts with Cr(VI) in an acidic solution to form a purplish-red complex that absorbs light at 540 nm. The addition of the ChrR+YieF solution that is mixed with Cr(VI) with reagents 1 and 2 results in an acidic solution with a purple-red color. The higher the rate of degradation of Cr(VI), the lighter the color of the tube is, since the concentration of Cr(VI) decreases as the production of Cr(III) forms(equilibrium).

This line graph demonstrates the trendline for the rate of Cr(VI) degradation by ChrR+YieF. It has a relatively constant slope, explaining its constant yet slow degradation rate.



Figure 64. Combined Graph of ChrR+YieF Protein's Cr(VI) Degradation Under Different Cr(VI) Concentrations

References:

Ackerley, D F et al. “Chromate-reducing properties of soluble flavoproteins from Pseudomonas putida and Escherichia coli.” Applied and environmental microbiology vol. 70,2 (2004): 873-82. doi:10.1128/AEM.70.2.873-882.2004

Apart from our basic and composite parts, we also created a hardware device in order to accommodate and utilize our genetically-modified bacteria to their greatest efficacy. The hardware device, which we named the Microbial Remediator (MR), was initially designed to optimize the costs, scalability, and functionality of the bacteria and various components. The MR now contains three main components: Detection, Degradation, Precipitation, and Filtration. The following details the functions of the three main components:

Figure 65. Diagram of the Hardware Device, Microbial Remediator, in (A) Models and (B) Real Assembly

• Connection with bacterial storage zones to fulfill Cr(VI) degradation purposes.
• A thorough mix of degradation bacteria in the sample fluid.
• Maintenance of bacterial survival in the Degradation Chamber.
• UV Disinfection of the degradation of E. coli bacteria.
• Transport of sample fluid into the Detection Chamber.
• Transport of sample fluid into the Precipitation and Filtration Chamber.

Figure 66. Close-Up Diagram of the Degradation Chamber

• Connection with bacterial storage zones to fulfill Cr(VI) detection purposes.
• A thorough mix of detection bacteria in the sample fluid.
• Maintenance of bacterial survival in the Detection Chamber.
• UV Disinfection for the detection of E. coli bacteria.
• Retrieval of fluid from the Degradation Chamber.
• Transport of fluid into the waste collection zone.

Figure 67. Close-Up Diagram of the Detection Chamber

• Retrieval of fluid from the Degradation Chamber.
• Addition of NaOH (aq) into the fluid.
• Thorough mix of NaOH (aq) in the fluid.
• pH detection of fluid to ensure no Cr(III) ions remain.
• Filtration of precipitate and dead E. coli out of the fluid.

Figure 68. Close-Up Diagram of the Precipitation and Filtration Chamber