Metallothionein (MT) was introduced into our plasmid backbone pASapI via Gibson assembly. This sequence was designed specifically with two type IIS restriction sites for SapI on either side of the MT sequence and ordered from IDT. This allowed us to utilize the MT plasmid as the backbone for all consecutive cloning and enabled us to proceed with golden gate assembly in the future. MT was cloned into 5-alpha competent E. coli cells from NEB. The transformation was successful, as the positive control plate for MT had significantly more colonies than the negative control plate. This cloning was confirmed via restriction digest using XbaI and BstxI. During the Gibson assembly, we excised the NcoI-HindIII linker in the pASapI backbone which contained an XbaI site. Therefore, the restriction digests of MT should only yield two bands (4.4kb, 2.4 kb) instead of the three bands from pASapI (4.4kb, 1.4kb, 0.8kb). The presence of only two bands, the increased size of the second band, indicated that MT was successfully cloned into the plasmid. For all future experiments, this plasmid was used as the backbone, known as MT-pASapI.
ArsC & FtnA
Arsenate reductase (ArsC) and ferritin (FtnA) were introduced separately into our plasmid backbone MT-pASapI via golden gate assembly. The constructs were designed by our team and produced by IDT. They were transformed into 5-alpha competent E. coli cells from NEB. The cloning was successful, as the positive control plate for ArsC and FtnA showed significantly more colonies than the negative control plates. This was confirmed with a restriction digest of the plasmid using XbaI and BstxI. FtnA was expected to have two bands: 4.4kb and 2.7 kb, while ArsC was expected to have two bands of 4.4kb and 2.6kb. This is a contrast to the MT-pASapI backbone, which would result in two bands of 4.4kb and 2.3kb 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.
ArsC-Ha, Mt-6xHIS, FtnA-Flag
The tagged sequences ArsC-Ha, MT-6xHIS, and FtnA-FLAG were introduced separately into our plasmid backbone MT-pASapI via golden gate assembly. The constructs were designed by our team and produced by IDT. They were transformed into 10-Beta competent E. coli cells from NEB. The cloning was successful, as there were more colonies on the positive control plates than on the negative controls. However, this was confirmed via restriction digest of the plasmid using BstXI and AflII. MT-6xHIS was expected to have 3 bands: 4.8kb, 1.3kb, and 809bp. ArsC-HA was expected to have two bands: 5.8kb and 1.3kb. FtnA-Flag was expected to have 2 bands: 5.9 kb and 1.3kb. 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.
MT-FtnA & MT-ArsC & ArsC-FtnA
The combinations MT-FtnA, MT-ArsC, and ArsC-FtnA were ultimately cloned into the MT-pASapI backbone via golden gate assembly. The constructs were designed by our team and produced by IDT. They were transformed into 10-Beta competent E. coli cells from NEB. The cloning was ultimately successful, after a few iterations and design changes. For more information about the iterative design process, see our Engineering Success page! The inserted sequences were confirmed via restriction digest using XbaI and BstxI. MT-FtnA digest was expected to have two bands of 4.4kb and 2.4kb. MT-FtnA was expected to have two bands of 4.4kb and 3.2kb. MT-ArsC was expected to have two bands of 4.4kb and 3.7kb. The number and size of bands shown in the gel were as expected, and indicated that the plasmid had been successfully integrated with our combined parts.
Full and Tagged
The “Full” sequence, MT-IEE2-ArsC-IEE5-FtnA, was designed by our team and produced in two parts by IDT. However, our team did not have any success cloning this construct via golden braid. This may be due to the presence of IEE2, which is also present in the pASapI backbone. For more information, see our Engineering Success page. We were unable to confirm cloning via restriction digest and often did not get significant results from the transformation into 10-beta competent E. coli cells.
Four rounds of transformation of C. reinhardtii were conducted in an attempt to transform a mutant strain of C. reinhardtii with our constructs. Three of the transformation attempts utilized the gene gun created by Dr. Redding. For this biolistic device, tungsten particles were coated in our constructs and shot at TBP plates coated with psbH deficient C. reinhardtii (CC-4388). These plates were then left to incubate under light for 2-4 weeks, and checked for small green colonies. Because the mutant C. reinhardtii is photosystem II deficient, the constant light source serves as selection criteria: only colonies that have integrated our plasmid with the rescue psbH gene will survive in the light conditions. The initial transformation, via gene gun, served as a positive control and learning session and yielded no results. The plasmid pASapI was the only construct tested. This failure was most likely due to human error, as it was the team’s first time using a biolistic device to transform microalgae. After 4 weeks under constant light exposure, no visible colonies were present, and the plates were discarded.
The second transformation utilized the same biolistic gene gun device/protocol and attempted to integrate ArsC, FtnA, MT, and pASapI (positive control) individually into the mutant CC-4388. This round of transformation was also unsuccessful, and no transformed colonies appeared. After 4 weeks under constant light exposure, no visible colonies were present, and the plates were discarded.
The third transformation utilized the same biolistic gene gun but incorporated a few modifications to the protocol. Most notably, we ensured that the vacuum in the chamber was sufficient, and introduced a ‘dark protocol’ at the suggestion of our peers on the Marburg team. They suggested we incubate the plates in the dark for 24 hours after shooting and then expose them to constant light for 2-4 weeks. To test this hypothesis, we shot four plates of each construct: plates 1 and 2 were placed directly in the light, and plates 3 and 4 were placed in the dark for ~24 hours and then exposed to the light for 4 weeks. This round of transformation yielded three transformed colonies after about 4 weeks of incubation in the light! The transformants were picked, struck out on TAP plates, and cultured for our experiments.
iGEMC1 - Single colony picked from MT plate #4 (Dark protocol)
iGEMC2 - Single colony picked from ArsC-HA plate #2 (Light protocol)
iGEMC3 - Single colony picked from MT-6xHIS plate #2 (Light protocol)
During the month that the third transformation was incubating in the light, we performed a glass bead transformation of CC-5168, a mutant of C. reinhardtii that is both psbH deficient and cell wall deficient. This protocol involved vortexing the algae with our plasmid and glass beads and plating it within top agar. While this protocol was simpler than the gene gun protocol, it did not yield any transformants.
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 our transformants, iGEMC1, iGEMC2, and iGEMC3, 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. This test revealed that photosystem II was successfully restored in all of our transformants. 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 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 . 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 graphs below, indicates that the rescue gene psbH from our plasmid pASapI was successfully localized into the chloroplast genome and restored photosynthetic function.
Polymerase chain reaction
PCR was run on extracted Chlamy genomic DNA from the successfully transformed constructs to confirm the integration of the rescue genes and recombinant proteins. These tests were 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 the constructs, we are still confirming the integration of our insertions for the final constructs.
ArsC-HA and MT-6xHIS, respectively. In order to determine if the arsenate reductase and metallothionein proteins are being expressed in the microalgae, we performed a western blot. The proteins were extracted from strains C2, C3, and WT C. reinhardtii, prepared for western blot, and loaded into the gel in duplicate. This allowed us to cut the membrane in half, enabling us to test for both the ArsC-HA protein and MT-6xHIS protein without stripping and re-tagging the same membrane. Both membranes were blocked in 5% milk TBST mixture for 30 mins. One half of the membrane, containing all three strains’ proteins, was tagged with a primary anti-HA tag at 1:1000 dilution and a secondary anti-rabbit HRP antibody at a dilution of 1:5000. The other half of the membrane, also containing all three strains’ proteins, was 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-HA antibodies was expected to show a 14.8kDa protein (ArsC) in well 3 (Image 1) . The membrane exposed to anti-6xHIS antibodies was expected to show a 6.5kDa protein (MT) in well 4 (Image 2) . 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-HA tagged membrane showed two bands in wells 2, 3, and 4 around and above the 25kDa mark (see image 1). This indicates that there is a protein present in both the wild type (well 2) and the engineered microalgae that expresses the HA tag. This does not definitively show the presence of the desired protein. In contrast, the anti-6xHIS tagged membrane was completely overexposed, even when the film was only placed on the membrane for 1-2 seconds (see image 2). 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.
Image 1: Anti-HA tagged membrane. Film was exposed for 2-5s. Wells L to R: Ladder, WT, C2, C3. “g” label on ladder indicates 25kDa marker.
Image 2: Anti-6xHIS tagged membrane. Film was exposed for 2-5s. Wells L to R: LAdder, WT, C2, C3. “G” Label on ladder indicates 25kDA marker.
The mutant C. reinhardtii strains iGEMC1, iGEMC2, and iGEMC3 were exposed to media contaminated with arsenic at 50ppb and 500 ppb for 48 and 72 hours to determine the arsenic sequestration rate. The strains were cultured, spun, down, and re-suspended in 40 mL of arsenic contaminated TP media at about 5E5 cells/ml. Three biological replicates were tested for each strain 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 via the Westerhoff lab at ASU. 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 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. There was also a statistically significant difference between the arsenic sequestration rate of the WT strain and the engineered C2 strain when exposed to 50 ppb contaminated media with p< 0.05. This difference is denoted in the box graph. This indicates that the engineered strains iGEMC2 and iGEMC3 sequester 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.
This image 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.
This image 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
This image 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.
- E.H. Murchie, T. Lawson, Chlorophyll fluorescence analysis: a guide to good practice and understanding some new applications, Journal of Experimental Botany, Volume 64, Issue 13, October 2013, Pages 3983–3998, https://doi.org/10.1093/jxb/ert208