Difference between revisions of "Part:BBa K4006004"

Latest revision as of 00:40, 22 October 2021

MT-6XHis

Background

This part contains a protein coding sequence for metallothionein that has been codon optimized for use in the chloroplast of Chlamydomonas reinhardtii as well as a 6X-histidine tag for visualization of protein expression in a Western blot. . 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. This version of metallothionein is codon optimized for use in the C. reinhardtii chloroplast and is an improved part, based off of the previously characterized part, BBa_K3275000, human metallothionein from team iGEM19_RHIT. The polyhistidine tag is well established and has been used previously in C. reinhardtii to tag the photosystem I complex.

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-6XHis into plasmid backbone MT-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.

MT-6xHIS was expected to have 3 bands: 4.8kb, 1.3kb, and 809bp. The original plasmid should have three bands of sizes 4446, 1376, and 800 bp. The number and size of bands revealed from the gel were as expected, indicating that the plasmid had been successfully integrated with our tagged inserts, with a few exceptions that were noted.

Figure 2. Gel electrophoresis of restriction digest with XbaI and BstXI of original pASapI plasmid and plasmid with integrated MT-6XHis. The gel showed three bands for all of the constructs run, the top band being undigested plasmid due to ineffective restriction digest. However, the other bands showed that the inserts contained bands of the appropriate size, and were different from the MT plasmid.

Transformation into Chlamydomonas reinhardtii

We were able to successfully transform this construct into C. reinhardtii and test integration, expression, 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 (C3) 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-6XHis (the yellow 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 C3, 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 iGEMC3 (MT-6XHis) 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-6XHis is represented as C3.

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 Expression of Proteins via Western Blot

In order to determine if the metallothionein protein is being expressed in the microalgae, we performed a western blot. The proteins were extracted from the algae, prepared for western blot, and loaded into the gel in duplicate. The membrane was blocked in 5% milk TBST mixture for 30 mins and tagged with a primary anti-6xHIS tag at 1:1000 dilution, and a secondary anti-mouse HRP antibody at a dilution of 1:1000. Imaging was performed in a dark room with film. Each membrane was covered in chemiluminescent reagent, and the film was exposed to the membrane for 2-10 seconds, then developed.

The membrane exposed to anti-6xHIS antibodies was expected to show a 6.5kDa protein (MT) in well 4. Despite our best efforts, the western blot performed did not provide any definitive results that the desired protein was being expressed in the engineered algae. The anti-6xHIS tagged membrane was completely overexposed, even when the film was only placed on the membrane for 1-2 seconds. This indicates a larger problem with some of the western blot. This membrane will be stripped and re-tagged with a lower concentration of secondary antibodies and re-imaged. In the future, another western blot should be performed.

Figure 6. The Western blot was overexposed and could not provide any reliable data.

Testing for Increased Arsenic Uptake Capabilities

The mutant C. reinhardtii strain iGEMC3 (MT-6XHis) 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. There was a statistically significant difference between the arsenic sequestration rate of the WT strain and the engineered C3 strain when exposed to 50ppb contaminated media with p< 0.05. This indicates that the engineered strain iGEMC3 sequesters arsenic at a faster rate than wild type algae, and would be more effective at reducing the arsenic contamination in ground water at concentrations of 50 ppb. 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 7. 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 (iGEMC3 is indicated by the yellow line.)

Figure 8. 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 (iGEMC3 is indicated by the yellow line.)

Figure 9. 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 (iGEMC3 is indicated by the green bar.)

Sequence and Features