Difference between revisions of "Part:BBa K4006005"

Line 2: Line 2:
 
<partinfo>BBa_K4006005 short</partinfo>
 
<partinfo>BBa_K4006005 short</partinfo>
  
This part contained a protein coding sequence for arsenate reductase that has been codon optimized for use in the chloroplast of Chlamydomonas reinhardtii as well as a human influenza hemagglutinin (HA) tag for visualization of protein expression in a Western blot. Arsenate reductase catalyzes the reduction of arsenate [As(V)] to arsenite [As(III)]. Using 2 µM arsenate at a substrate, the Kcat of the protein has been recorded as 4.5 min(-1).
+
===Background===
  
We were able to clone this construct into our plasmid and select for transformed E. coli colonies.
+
This part contains a protein coding sequence for arsenate reductase that has been codon optimized for use in the chloroplast of ''Chlamydomonas reinhardtii'' as well as a human influenza hemagglutinin (HA) tag for visualization of protein expression in a Western blot. Arsenate reductase catalyzes the reduction of arsenate [As(V)] to arsenite [As(III)]. Using 2 µM arsenate at a substrate, the Kcat of the protein has been recorded as 4.5 min(-1). This version of arsenate reductase is codon optimized for use in the ''C. reinhardtii'' chloroplast and is a new part.
  
PLATE PICTURES
+
===Cloning into ''E. coli'' and Verification===
  
Digestion of the miniprepped DNA in the plasmid pASapI with XbaI and BstXI should result in two bands of approximate sizes SIZE bp and SIZE 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.
+
We were able to clone this construct into our plasmid, pASapI, using Gibson assembly and select for transformed ''E. coli'' colonies.
  
GEL PICTURES
+
[[Image:T--ASU--MT6Xplates.png|center|thumb|700px|<b>Figure 1.</b> The plate on the left is the experimental transformation of arsC-HA 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.]]
  
We were able to successfully transform this construct into C. reinhardtii using biolistic transformation. First, we used SOMETHING ABOUT LIGHT, EMMA?? to confirm that the photosystem II subunit had been restored with our rescue plasmid.
+
ArsC-HA was expected to have two bands: 5.8kb and 1.3kb. 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 the second well being a noted exception.
  
RESULTS OF LIGHT THING
+
[[Image:T--ASU--MT6Xgel.png|center|thumb|500px|<b>Figure 2.</b> Gel electrophoresis of restriction digest with XbaI and BstXI of original pASapI plasmid and plasmid with integrated arsC-HA. 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.]]
  
Next, we performed PCR using primers outside of the region where our protein was meant to integrate into the genome. JARED SOMETHING ABOUT RESULTS.
+
===Transformation into ''Chlamydomonas reinhardtii''===
  
IMAGES
+
We were able to successfully transform this construct into ''C. reinhardtii'' and test integration, expression, localization, and effect on arsenic uptake in the algae.
  
The HA tag is also generally well established and has been used previously in C. reinhardtii to tag putative flagellar proteins. Interestingly, though, when we used anti-HA antibodies in a Western blot for our transformant colony's genomic DNA, we visualized two distinct bands present throughout all of our constructs and the wildtype. They were not the right size for the arsC, yet we have seen no literature reporting on the natural presence of the HA sequence within the C. reinhardtii genome.
+
===Testing for Integration of Plasmid via Fluorescence with Spectrophotometry===
  
PICTURES OF BLOTS.
+
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 (C2) 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.
  
 +
[[Image:T--ASU--FluorLine.png|center|thumb|600px|<b>Figure 3.</b> Maximum quantum yield of photosystem subunit II. arsC-HA (the red line) indicates a peak in fluorescence, as opposed to the lack of a peak in the wildtype.]]
  
<!-- Add more about the biology of this part here
+
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 C2, 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.
===Usage and Biology===
+
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.
  
<!-- -->
+
[[Image:T--ASU--FluorBar.png|center|thumb|600px|<b>Figure 4.</b> Calculation of Fo/Fm and comparison of values between different strains and mutated wildtype lacking photosystem II. Each of the strains, specifically iGEMC2 (arsC-HA) approached the photosynthetic efficiency of that of the wildtype strain, indicating restoration of the subunit by integration of our plasmid.]]
<span class='h3bb'>Sequence and Features</span>
+
<partinfo>BBa_K4006004 SequenceAndFeatures</partinfo>
+
  
 +
===Testing for Localization of Plasmid via PCR===
  
<!-- Uncomment this to enable Functional Parameter display
+
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, arsC-HA is represented as C2.
===Functional Parameters===
+
 
<partinfo>BBa_K4006004 parameters</partinfo>
+
[[Image:T--ASU--PCR.png|center|thumb|400px|<b>Figure 5.</b> 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===
 +
 
 +
NEED THIS BLURB AND IMAGE
 +
 
 +
===Testing for Increased Arsenic Uptake Capabilities===
 +
 
 +
The mutant C. reinhardtii strain iGEMC2 (arsC-HA) 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 C2 strain when exposed to 50ppb contaminated media with p< 0.05. This indicates that the engineered strain iGEMC2 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.
 +
 
 +
[[Image:T--ASU--ArsenicPlot50.png|center|thumb|600px|<b>Figure 6.</b> 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. Tagged arsenate reductase (iGEMC2 is indicated by the red line.)]]
 +
 
 +
 
 +
[[Image:T--ASU--ArsenicPlot500.png|center|thumb|600px|<b>Figure 7.</b> 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. Tagged arsenate reductase (iGEMC2 is indicated by the red line.)]]
 +
 
 +
 
 +
[[Image:T--ASU--ArsenicBar.png|center|thumb|600px|<b>Figure 8.</b> 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. Tagged arsenate reductase (iGEMC2 is indicated by the orange bar.)]]
 +
 
 +
 
 +
<span class='h3bb'>Sequence and Features</span>
 +
<partinfo>BBa_K4006005 SequenceAndFeatures</partinfo>

Revision as of 21:57, 21 October 2021

arsC-HA

Background

This part contains a protein coding sequence for arsenate reductase that has been codon optimized for use in the chloroplast of Chlamydomonas reinhardtii as well as a human influenza hemagglutinin (HA) tag for visualization of protein expression in a Western blot. Arsenate reductase catalyzes the reduction of arsenate [As(V)] to arsenite [As(III)]. Using 2 µM arsenate at a substrate, the Kcat of the protein has been recorded as 4.5 min(-1). This version of arsenate reductase is codon optimized for use in the C. reinhardtii chloroplast and is a new part.

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 arsC-HA 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.

ArsC-HA was expected to have two bands: 5.8kb and 1.3kb. 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 the second well being a noted exception.

Figure 2. Gel electrophoresis of restriction digest with XbaI and BstXI of original pASapI plasmid and plasmid with integrated arsC-HA. 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 (C2) 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. arsC-HA (the red 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 C2, 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 iGEMC2 (arsC-HA) 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, arsC-HA is represented as C2.

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

NEED THIS BLURB AND IMAGE

Testing for Increased Arsenic Uptake Capabilities

The mutant C. reinhardtii strain iGEMC2 (arsC-HA) 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 C2 strain when exposed to 50ppb contaminated media with p< 0.05. This indicates that the engineered strain iGEMC2 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 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. Tagged arsenate reductase (iGEMC2 is indicated by the red 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. Tagged arsenate reductase (iGEMC2 is indicated by the red 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. Tagged arsenate reductase (iGEMC2 is indicated by the orange bar.)


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 5
    Illegal SapI.rc site found at 470