Latest revision as of 21:08, 21 October 2021

Arsenic metallothionein

Human arsenic targeting metallothionein

Sequence and Features

Introduction

Metallothionein (MT) is a class of small metal-binding proteins that exists in bacteria, plants and animals. These proteins depending on their amino acid compositions have a high binding affinity with different bivalent metal ions. Once MT detects the corresponding metal, it binds the goal through covalent bonds, which are composed of sulfhydryl cysteine residues and stores the metal by tightly chelating the metal. Typically, it is assumed that MTs have two binding domains, one of which is the C-terminal part (α-domain) with three binding sites. The other one is the N-terminal part (β-domain) with four divalent binding sites ^[1]. Therefore, MTs are important for protecting the cell against heavy metal toxicity and maintaining cellular homeostasis.

Arsenic Metallothionein

As(V) can be reduced to As(III) by arsenate reductase, and then the arsenite can bind to thiol groups easily ^[2].Metallothionein is a great tool for E.coli to accumulate arsenic because it has a great amount of cysteine, which makes it a thiol-rich protein. part:BBa_K3275000 is from human metallothionein originally, which is called Metallothionein-1A (MT-1A). There are two metal binding domains in MT-1A: the α domain and the β domain. In the α domain, cysteinyl thiolate bridges let 11 cysteine ligands to coordinate with 4 divalent ions, and these ions are chelated with cluster A. In the β domain, the corresponding region, cluster B, helps ligate 3 divalent ions to 9 cysteines ^[3]. Figure 1 shows the structure of MT-1A ^[4]. The figure shows that MT-1A can bind with 2 zinc ions and 5 cadmium ions.

Figure 1. 3-D structure of MT-1A find more here

For part:BBa_K3275000, some bases from the original sequence are changed due to synthesis demands.

Characterization

The culmination of testing for this project was to examine how well both constructs (A1 and A2) could handle the bioremediation of arsenic. The protocol can be found in the last second page of the Protocol section in the RHIT|Experimentor in the RHIT|Notebook Details on the parts used in the design section of the wiki. Our results show that both A1 and A2 were successful at removing arsenite ions from the aqueous solution, and as the total amount of arsenite continued to move down as time increased, cells were able to survive as well. Small decreases were noticed in the negative control as well, as arsenite is naturally transported in and out of cells through aquaglyceroporins (AQPs). In the shorter term (8 hours), A2, which produces metallothionein constantly due to its constitutive promoter, showed better bioremediation of the arsenite. However, at the longer time scale, the arsenic controlled (through ArsR) A1 ended up proving more effective at removing arsenic.

Codon Optimization for Use in Chlamydomonas reinhardtii by iGEM ASU 2021

We have codon optimized this part for use in the chloroplast of Chlamydomonas reinhardtii. The sequence of our new part is available at BBa_K4006002 Metallothionein is a metal binding protein rich in cysteine that is commonly produced in bacteria and prokaryotes. It has been introduced to the Chlamydomonas genome before and significantly improves the metal binding capacity at low metal concentrations.

Cloning into E. coli and Verification

We were able to clone this construct into our plasmid, pASapI, using Gibson assembly and select for transformed E. coli colonies.

Figure 1. The plate on the left is the experimental transformation of MT into plasmid pASapI using Gibson Assembly. Multiple individual colonies are present on the plate, indicating successful transformation. The plate on the right is the negative control. Little to no colonies are present on the plate, indicating that there should not be high background or incomplete recombination in our experimental plate.

Digestion of the miniprepped DNA in the plasmid pASapI with XbaI and BstXI should result in two bands of approximate sizes 4446 bp and 2332 bp as compared to the original plasmid which should have three bands of sizes 4446, 1376, and 800 bp. Each of the colonies were successfully cloned.

Figure 2. Gel electrophoresis of restriction digest with XbaI and BstXI of original pASapI plasmid and plasmid with integrated MT. Each of the picked colonies (MT 1A-1D) indicate two distinct bands as compared to the pASapI's three bands. The largest band on the pASapI is highly visible, but the other two are much more subtle.

Transformation into Chlamydomonas reinhardtii

We were able to successfully transform this construct into C. reinhardtii and test integration, localization, and effect on arsenic uptake in the algae.

Testing for Integration of Plasmid via Fluorescence with Spectrophotometry

We performed a fluorescence test to determine if the rescue gene psbH in our plasmid successfully localized to the chloroplast genome and restored function of photosystem II. This test utilized a Joliot kinetic spectrophotometer to measure the maximum quantum yield of photosystem II (PSII). We tested this construct (C1) among others, as well as the wild type strain CC-124 and the photosystem II deficient mutant, CC-4388 as a positive and negative control, respectively. To determine the maximum quantum yield of photosystem II, a bright flash of blue saturating light is emitted from the spectrophotometer, and the resultant red light fluorescence from the microalgae is measured. The fluorescence, as seen in the line graph below, was normalized to the background fluorescence emitted by an empty TBP plate.

Figure 3. Maximum quantum yield of photosystem subunit II. MT (the blue line) indicates a peak in fluorescence, as opposed to the lack of a peak in the wildtype.

This test revealed that photosystem II was successfully restored in this transformant. The wild type strain, CC-124, shows an increase in emitted fluorescence when the saturating flash is emitted (green line). All three of the transformants, but specifically C1, show a similar peak (blue, orange, and yellow lines), indicating that photosystem II function has been restored. In contrast, the mutant CC-4388, which is PSII deficient, does not show any peak in fluorescence (gray line). The difference in the size of the peaks is due to differing concentrations of algae on the plates that were measured. To normalize the data to each algae’s relative density on the plate, the Maximum quantum efficiency of PSII photochemistry was calculated using the equation (Fm-Fo)/Fm [1]. The baseline fluorescence, known as Fo, is the amount of fluorescence measured emitting from the algae before the saturating flash. The maximum fluorescence signal, Fm, is measured at the peak of the saturating flash. Normalizing this data reveals how much the fluorescence changed when exposed to the bright saturating flash. The wild type strain, CC-124, had an Fv/Fm ratio of 0.8. All three of the mutant strains very nearly approached this ratio as well (0.756, 0.747, and 0.761, respectively for C1,C2, and C3). However, the photo deficient mutant CC-4388 had an Fv/Fm value of 0.008, indicating that there was no PSII activity. This data, as represented by the graph below, indicates that the rescue gene psbH from our plasmid pASapI was successfully localized into the chloroplast genome and restored photosynthetic function.

Figure 4. Calculation of Fo/Fm and comparison of values between different strains and mutated wildtype lacking photosystem II. Each of the strains, specifically iGEMC1 (MT) approached the photosynthetic efficiency of that of the wildtype strain, indicating restoration of the subunit by integration of our plasmid.

Testing for Localization of Plasmid via PCR

PCR was run on extracted Chlamy genomic DNA from the successfully transformed construct to confirm the integration of the rescue genes and recombinant proteins. This was performed to confirm integration in the chloroplast genome, as the rescue system is active when integrated into the chloroplast genome. We designed primers according to the deletion strain (CC-4388) sequence. Theoretically, these primers are capable of binding to the rescue gene flanking sites approximately 50bp upstream and downstream of the integration site. This can effectively amplify the site which will confirm that the plasmid donor DNA has effectively been integrated, and that the insertion is of approximately accurate size. Attempts with multiple polymerases and annealing temperatures proved unsuccessful at targeting the region of interest, indicating that the likely problem resides in the effective binding accuracy of the primers. Further review confirmed that the sequence targeted may have slight adjustments based on a current review of genomic sequencing. While we were able to effectively extract genomic information from the wild type strain and this construct, we are still confirming the integration of our insertions for the final constructs. In the image below, MT is represented as C1.

Figure 5. The agarose gel confirms that there is genomic DNA present in our samples, but we were unable to isolate the region of interest using PCR. This may be due to the binding accuracy of our designed primers or size of the region of interest.

Testing for Increased Arsenic Uptake Capabilities

The mutant C. reinhardtii strain iGEMC1 (MT) was exposed to media contaminated with arsenic at 50ppb and 500 ppb for 48 and 72 hours to determine the arsenic sequestration rate. The strain was cultured, spun, down, and re-suspended in 40 mL of arsenic contaminated TP media at about 5E5 cells/ml. Three biological replicates were tested at each concentration. Samples were collected at 0 hrs, 48 hrs, and 72 hrs, and the cell count of each sample was measured at that time. The arsenic concentration in the supernatant was measured using ICP-MS analysis.

In order to normalize the data to the growth rate of each sample, the sequestration rate was calculated in terms of arsenic concentration per fold growth of algae. This accounts for the increased arsenic uptake that may occur when the algae is more prolific. As seen in the two images below, the arsenic sequestration rate of all of the mutant strains is higher than that of the wild type algae. This is true for both 50ppb arsenic concentrations as well as 500ppb arsenic concentrations. In both graphs below, the wild type CC-124 strain arsenic sequestration rate is shown in green. The slope of this line (trendline through all three biological replicates) has a slope of -26.5 for 50ppb, and -321.4 for 500 ppb. Both of these values are smaller than those of the engineered strains. However, an ANOVA test was performed to determine if this difference was statistically significant. While there were significant differences with other constructs, MT (C1) did not show a signficant difference. There was no significant difference in the sequestration rate for any of the algae exposed to 500 ppb media. This is most likely because the amount of arsenic the cells can sequester is small in comparison to such a high concentration of arsenic. This merits further exploration and testing.

Figure 6. This graph shows the normalized arsenic sequestration rate of the engineered constructs iGEMC1, iGEMC2, iGEMC3, and the wild type, WT, when exposed to 50 ppb of arsenic. Metallothionein (iGEMC1 is indicated by the blue line.)

Figure 7. This graph shows the normalized arsenic sequestration rate of the engineered constructs iGEMC1, iGEMC2, iGEMC3, and the wild type, WT, when exposed to 500 ppb of arsenic. Metallothionein (iGEMC1 is indicated by the blue line.)

Figure 8. This graph compares the normalized arsenic sequestration rate (slopes) of the engineered constructs iGEMC1, iGEMC2, iGEMC3, and the wild type, WT, at 50 ppb, and indicates statistically significant difference between WT/C3 at 50 ppb and WT/C2 at 50 ppb. Metallothionein (iGEMC1 is indicated by the blue line.)

References

1. ↑ Ruttkay-Nedecky, B., Nejdl, L., Gumulec, J., Zitka, O., Masarik, M., Eckschlager, T., … Kizek, R. (2013). The role of metallothionein in oxidative stress. International journal of molecular sciences, 14(3), 6044–6066. doi:10.3390/ijms14036044
2. ↑ Ngu, T. and Stillman, M. (2006). Arsenic Binding to Human Metallothionein. Journal of the American Chemical Society, 128(38), pp.12473-12483.
3. ↑ MT1A - Metallothionein-1A - Homo sapiens (Human) - MT1A gene & protein. (2019). Retrieved from https://www.uniprot.org/uniprot/P04731
4. ↑ SWISS-MODEL Repository | P04731. (2019). Retrieved from https://swissmodel.expasy.org/repository/uniprot/P04731?csm=8FBA7C54EE8B6A13