The biomarker Calprotectin consists of protein subunits S100A8 and S100A9 that can bind to zinc ions (Zn2+). It is related to diseases like rheumatoid arthritis, systemic lupus erythematosus, and inflammatory bowel disease.In order to construct an E. coli that can detect the biomarker Calprotectin, we adopted the Two-Component System(TCS) SaeRS from Staphylococcus aureus.
The SaeRS system of Staphylococcus aureus is composed of two proteins, SaeR and SaeS, which work together to detect environmental signals and regulate downstream gene expression. When the sensor histidine kinase SaeS phosphorylates the response regulator SaeR, the phosphorylated SaeR binds to a direct repeat sequence in specific downstream promoters, activating gene expressions. However, in the presence of Zn2+, the phosphorylation of SaeS is inhibited, preventing the phosphorylation of SaeR and consequently inhibiting downstream gene expression. There are studies that show when Calprotectin binds Zn2+, it could effectively prevent downstream gene expressions from being inhibited.[1] This implies that when Calprotectin is present, it would increase downstream gene expression.
We apply this mechanism to our experiment design. Phla is the promoter of the hla gene in Staphylococcus aureus. It is one of the promoters that is regulated by the two-component system SaeRS. The experiment concept we adopted is that under the presence of a higher concentration of Calprotectin, there would be fewer Zn2+ free ions. With fewer Zn2+ free ions, the expression of the gene for the fluorescent protein mCerulean would increase, producing a higher fluorescent intensity for us to detect. This way, we can infer the concentration of Calprotectin by observing the fluorescent density emitted by the E. coli.
We retrieved the plasmids for the promoter phla (pYJ-Phla-lacZ plasmid) and the two-component gene saeRS (pCL-sae-usa plasmid) from Professor Taeok Bae from the Department of Microbiology and Immunology, Indiana University School of Medicine-Northwest, USA. We transformed the two plasmids into E. coli DH5ɑ, respectively, to amplify them. The bacteria were cultured in LB broth with antibiotic Ampicillin. We extracted the aforementioned two plasmids which contain the phla promoter and the gene saeRS, and we also extracted plasmids that contained the gene for the fluorescent protein mCerulean.
We used Polymerase Chain Reaction (PCR) to amplify specific DNA sequences we needed for the new plasmid DNA construction. These gene fragments include gene saeRS, promoter p3, promoter phla, and the gene for mCerulean. In the beginning, we used the typical PCR programs for each fragment, but the amplification results were unsuccessful after several tries. Therefore, we conducted gradient PCR to discover the favorable temperature condition. We employed the adjusted program settings (see more at Protocol) based on the gradient PCR results and ultimately obtained the desired PCR products. Later, electrophoresis was done to verify each DNA fragment length and we conducted gel extraction to retrieve them for further experiments.
We ligated the five genes, saeRS, Terminator, Origin+CmR, phla, and the gene for mCerulean, for our construct design using Gibson Assembly and transformed the engineered plasmid into E. coli. We adjusted the proportion of each part until we succeeded in cloning our genes into an assembled plasmid.
To ensure that we have successfully assembled all parts of our genes and that the E. coli can express the genes of interest, we extracted the proteins that were produced by the E. coli and performed Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis(SDS-PAGE).
After the first SDS-PAGE result, we observed that the concentration of SaeS is extremely low. Therefore, we designed another plasmid, on which the promoter of saeRS is BBa_J23100, a constitutive promoter with high transcription levels. Meanwhile, we also plan to point mutate on the XbaI digestion site, and add a His tag on SaeR.
To determine the sensitivity of our engineered E. coli, we designed a functional assay to test it out. We cultured the engineered E. coli in various concentrations of Zn2+ and measured the fluorescent intensity and OD600 of the E. coli. The observed fluorescent density of the E. coli allowed us to assess the relationship between Zn2+ concentration and the expression level of the fluorescent genes..
L-Tyrosine is an essential amino acid in our bodies and it is associated with inflammation in patients with Lewy Body Dementia (LBD). In order to detect the presence of L-Tyrosine with our engineered E. coli, we introduced the usage of TyrR protein, a DNA-binding transcriptional dual regulator. The gene tyrR exists in E. coli K-12 MG1655, allowing it to produce the TyrR protein. TyrR has several binding sites for multiple substrates and could act as an activator or a repressor under different circumstances. In the presence of L-Tyrosine, TyrR can self-associate into hexamers that bind to both the strong box and weak box of the promoter, thereby influencing downstream gene expressions.
The promoter we selected was paroF, which is repressed in the presence of L-Tyrosine. Two TyrR protein hexamers are involved in this reaction. One of the TyrR hexamers can identify the TyrR box 1(strong box) and TyrR box 2(weak box), and then binding across them. Another hexamer binded to the upstream TyrR box 3(strong box) and the adjacent region.
Following the promoter paroF is the gene encoding the fluorescent protein mRFP1. The fluorescence emission intensity could indicate the degree of paroF repression and, subsequentially, the concentration of L-Tyrosine.
We received the E. coli carrying the pLacUV5-tyrR-paroF-mRFP1 plasmid from Professor Yi-Chun Yeh from the Department of Chemistry, National Taiwan Normal University, Taiwan. However, this plasmid bears the promoter pLacUV5, which is an unnecessary inducible promoter for our experiment design. Therefore, we decided to replace it with a constitutive promoter.
The pET-21a(+) plasmid was selected as the new backbone of tyrR-paroF-mRFP1 due to the presence of the constitutive T7 promoter in its sequence. It was then transformed into E.coli DH5ɑ and cultured on LB + Ampicillin agar plates. Colonies were picked and cultured in LB broth containing the antibiotic Ampicillin to confirm the success of the transformation.
Both the pLacUV5-tyrR-paroF-mRFP1 plasmid and the pET-21a(+) plasmid were digested with EcoRI and XhoI enzymes, followed by electrophoresis and gel extraction to obtain the desired DNA fragments. For PLacUV5-tyrR-ParoF-mRFP1 plasmid, the desired length would be 2496 base pairs, while for pET-21a(+) plasmid, the correct length would be 5409 base pairs.
We ligated both DNA fragments with T4 DNA ligase and left at room temperature for one hour. We tried two molar ratios of vector DNA to insert DNA, specifically 1:3 and 1:1, to determine a better recipe for DNA ligation. The ligated product was then transformed into E. coli DH5ɑ and cultured on LB + Ampicillin agar plate.
The engineered E.coli bacteria were transferred from LB + Ampicillin broth to M9 minimal broth, which was supplemented with a 0.4% glucose solution, 2 mM MgSO4 solution, 0.1 mM CaCl2 solution, 0.5 μg/mL Thiamine hydrochloride, and 100 μg/mL Ampicillin. The L-Tyrosine concentration was varied in a gradient from 0 μM to 100 μM, with intervals of 10 μM.
We measured the fluorescent intensity and OD600 from the engineered E. coli to verify the function of our detection device. The fluorescent density of Tyrosine biosensor decreased as the L-Tyrosine concentration increased. We concluded that our biosensor has the ability to detect the existence of L-Tyrosine. In the end, we examined whether our constructed biosensor could provide a rough estimate of the L-Tyrosine concentration in the supplemented M9 minimal broth by analyzing the fluorescent density data.
MtrA, MtrB, and MtrC are the major c-type cytochromes from Shewanella oneidensis involved in the extracellular electron transfer (EET) pathway, serving as electron carriers and helping the electron transfer. The CcmA-H proteins are necessary for the maturation of MtrA, MtrB, and MtrC cytochromes. In order to construct an EET pathway as one of the reporting systems in our project, we need to co-transform the pSB1C3-phla-(MtrCAB+tag)-Sae plasmid and the pYJ_BBa_J23100_CcmAH_BBa_B0015 plasmid which carries the gene ccmA-H into E. coli. [5][6]
We purchased the pETSXM2-pLacI-mtrCAB plasmid from Addgene. To connect with the upstream biomarker detection system, we replaced the mCerulean gene on the saeRS plasmid with the mtrCAB on the plasmid pETSXM2-pLacI-mtrCAB. We used PCR to amplify the mtrCAB part and added one of two restriction enzyme cutting sites, SacII and BamHI, to each end. Then, we used enzyme digestion to obtain the mtrCAB fragment and saeRS plasmid excluding the mCerulean gene. We ligated these two fragments together and completed pSB1C3-phla-(MtrCAB+tag)-Sae plasmid construction. (see more at Protocol)
The ccmA-H gene on the pYJ_BBa_J23100_CcmAH_BBa_B0015 plasmid is necessary for cytochrome maturation. Cytochrome maturation is a post-translational process involving covalent attachment of heme to the apocytochrome polypeptide by two thioether bonds. We extracted the gDNA of E. coli K-12 MG1655 and used PCR technique to amplify the ccmA-H part of the gDNA. Then we conducted enzyme digestion and ligation to combine ccmA-H genes with the replication origins, the Ampicillin resistant gene, the promoter BBa_J23100, the ribosome binding site BBa_B0034, and the terminator from the pSB1C3-mCerulean plasmid.
After we completed the construction of both plasmids above, we had to co-transform them into E. coli strain BL21 with the aim of obtaining expression strain.
To check if our constructed genes are expressed as expected, we induced E. coli to produce the protein, and then tested their performance properties. In the previous primer design, we added 6x his tag after our target genes mtrCAB and ccmA-H(we only added his tag after the mtrB and ccmH gene due to other genes being connected to each other). Thus, there will be 6 histidine at the end of the two proteins. Since histidine has a high affinity to the metal ion, Ni2+, we used Ni-column to catch the proteins with histidine with the aim to purify the protein product. (see more at Protocol)
After we purified the protein product, we needed to check whether the protein's properties were as we expected. We know from reviewing databases that the molecular weight of the MtrB protein is about 77kDa, and the CcmH protein is about 39kDa. By running SDS-Page, we can measure the mass of the protein and know if the E. coli can successfully synthesize the desired protein.(see more at Protocol)
To check if our cytochrome, MtrA, MtrB and MtrC, are being synthesized in the engineered E.coli, we conducted heme staining, which is an additional staining of SDS-Page. The gel is immersed in the 3,3',5,5'-Tetramethylbenzidine (TMBZ) solution, where the TMBZ will bind to the heme in the cytochromes. After adding hydrogen peroxide into the solution, the TMBZ-bound heme will turn bluish-green (λmax: 655 nm), therefore allowing us to see the cytochromes. (see more at Protocol)
We built up a simple device to check the functionality and feasibility of our engineered E. coli. We injected the engineered E. coli into a CoverWell Perfusion Chamber. We utilized the three-electrode system which consisted of two carbon electrodes, a working electrode and a counter electrode, one silver electrode as a reference electrode, and cyclic voltammetry to test the current intensity. For more details, see the dry lab device page. [7]
Hesperetin is a flavonoid compound, commonly found in citrus fruits, particularly in the peels of oranges, grapefruits, and lemons. It has many functions that benefit our health, like antioxidants, anti-inflammatory, anticancer, lipid-lowering, antidiabetic, etc. It can be produced by cleaving rhamnose and glucose glycosides from hesperidin by α-Rhamnosidase and β-Glucosidase.
Typically, humans absorb the precursor hesperidin and convert it into hesperetin using α-Rhamnosidase and β-Glucosidase enzymes within our bodies. However, the challenge for our bodies is that the amount of α-Rhamnosidase in our bodies varies among individuals. Even if someone consumes a lot of hesperidin, they may not be able to synthesize a comparable amount of hesperetin. And because α-Rhamnosidase is the rate-determining step in the reaction, which transforms hesperidin transfers to hesperetin, we chose to design the gene for α-Rhamnosidase into our engineered plasmid. The ultimate goal is to raise the quantity of α-Rhamnosidase in our bodies and help relieve inflammation responses.
α-Rhamnosidase is an enzyme responsible for cleaving rhamnose from hesperidin. The bacteria Streptomyces avermitilis carries the Rhamnosidase gene in its genomic DNA. Therefore, we employed this conversion method for this specific part of our project.
We conducted PCR to amplify the Rhamnosidase gene sequence. Additionally, by adding a His tag to the sequence, it is more convenient for us to identify the rhamnosidase protein in the subsequent experiments.
We used the pSB1C3 plasmid as the backbone in our plasmid construct. We employed the restriction enzyme sites HindIII and BamHI to insert the gene for the Rhamnosidase into our plasmid. We transferred the engineered plasmid into E. coli DH5α to amplify the plasmid. We cultured the E. coli in LB + Cm to confirm the success of the transformation.
We transformed the plasmid into E. coli BL21 to express Rhamnosidase.
We Used Ni-NTA to purify protein, then ran the SDS-PAGE to check if the E. coli expresses the Rhamnosidase protein.
We conducted in vitro experiments, applying the extracted Rhamnosidase to facilitate the conversion of hesperidin to hesperetin.
The objective of this experiment is to evaluate the conversion efficiency of hesperidin after feeding mice with E.coli containing the Rhamnosidase gene and hesperidin simultaneously. We plan to analyze the levels of hesperetin and hesperidin in their blood.
+886-2-2826-7000
nycu.taipei.igem@gmail.com
© 2023 - Content on this site is licensed under a Creative Commons Attribution 4.0 International license.
The repository used to create this website is available at gitlab.igem.org/2023/nycu-taipei.
+886-2-2826-7000
nycu.taipei.igem@gmail.com
© 2023 - Content on this site is licensed under a Creative Commons Attribution 4.0 International license.
The repository used to create this website is available at gitlab.igem.org/2023/nycu-taipei.