iGEM 2018 — Team ASIJ_Tokyo

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# Team:ASIJ Tokyo/Notebook

* [HOME](https://2018.igem.org/Team%3AASIJ_Tokyo)
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* [PROJECT](https://2018.igem.org/Team%3AASIJ_Tokyo/Description)
  + [CRISPR](https://2018.igem.org/Team%3AASIJ_Tokyo/CRISPR)
  + [DESIGN](https://2018.igem.org/Team%3AASIJ_Tokyo/Design)
  + [EXPERIMENT](https://2018.igem.org/Team%3AASIJ_Tokyo/Experiments)
  + [NOTEBOOK](https://2018.igem.org/Team%3AASIJ_Tokyo/Notebook)
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  + [DEMONSTRATE](https://2018.igem.org/Team%3AASIJ_Tokyo/Demonstrate)
* [PARTS](https://2018.igem.org/Team%3AASIJ_Tokyo/Parts)
  + [IMPROVE](https://2018.igem.org/Team%3AASIJ_Tokyo/Improve)
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  + [OUR ACTIVITIES](https://2018.igem.org/Team%3AASIJ_Tokyo/Activities)
  + [Q&A](https://2018.igem.org/Team%3AASIJ_Tokyo/Questions)
  + [FUTURE](https://2018.igem.org/Team%3AASIJ_Tokyo/Future)* [SAFETY](https://2018.igem.org/Team%3AASIJ_Tokyo/Safety)

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# NOTEBOOK

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# Team:ASIJ Tokyo/Experiments

* [HOME](https://2018.igem.org/Team%3AASIJ_Tokyo)
* [TEAM](https://2018.igem.org/Team%3AASIJ_Tokyo/Team)
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* [PROJECT](https://2018.igem.org/Team%3AASIJ_Tokyo/Description)
  + [CRISPR](https://2018.igem.org/Team%3AASIJ_Tokyo/CRISPR)
  + [DESIGN](https://2018.igem.org/Team%3AASIJ_Tokyo/Design)
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  + [NOTEBOOK](https://2018.igem.org/Team%3AASIJ_Tokyo/Notebook)
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* [PARTS](https://2018.igem.org/Team%3AASIJ_Tokyo/Parts)
  + [IMPROVE](https://2018.igem.org/Team%3AASIJ_Tokyo/Improve)
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# EXPERIMENTS

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#### Part I : [CRISPR](https://static.igem.org/mediawiki/2018/c/cc/T--ASIJ_Tokyo--crisprpart1.pdf) Part II : [CRISPR Confirmation (genotypic)](https://static.igem.org/mediawiki/2018/3/30/T--ASIJ_Tokyo--crisprpart2.pdf) Part III: Designing of the new constructs Part IV: [Gibson Assembly of Constructs](https://static.igem.org/mediawiki/2018/f/fd/T--ASIJ_Tokyo--gibson.pdf) Part V: [Behavior Control Lab Protocol](https://static.igem.org/mediawiki/2018/4/47/T--ASIJ_Tokyo--bevaviorcontrol.pdf) * To confirm our hypothesis regarding the secretion behavior of A1AT in cells as it differs between the M allele and ZZ allele we conducted a behavior control lab. Through our research, we discovered that antitrypsin of the SERPINA1 E342K (ZZ allele) with point mutation variant polymerizes within the cell, thus resulting in a greater cell mass as the protein is unable to be excreted. A protein of the SERPINA1 (MM) variant will successfully exit the cell, therefore resulting in lesser cell mass. To observe this behavior in our own scenario, each construct was tagged with a distinctly different colored fluorescence reporter, enabling us to see a qualitative difference in where the fluorescence, and therefore the protein, was located. In our experiment, the pattern in the location of fluorescence, due to the mass of the cells, followed the expected behavior. We also tried different fluorescence markers and compared the scenarios of an unmutated protein and a protein we conducted CRISPR on. The completion of the behavior control lab confirmed our hypothesis, provided a qualitative confirmation of the mutation’s successful correction, and decided the fluorescence marker used throughout the rest of the expirement.. Part VI: Genotypic and Phenotypic Proof of Concept * a. Agarose gel electrophoresis of plasmid constructs (agarose gel electorphoresis protocol) + We conducted the electrophoresis as a way to collect more evidence to establish that the CRISPR edited SERPINA1 E342K with point mutation construct was edited successfully and determine if the proteins produced behaved like the unmutated A1AT. The mutated construct, prone to polymerization is expected to have a larger weight, and thus travel less in the gel. On the other hand, the unmutated construct, with no polymerization, is expected to have a lighter molecular weight and thus travel farther in the gel. This method was chosen because the materials were readily available to us and many members of the team have performed it before. * b. Phenotypic Proof (Transformation and growing on selection plates) Part VII: Quantifying Protein * a. [GFP protein purification using column chromatography](https://static.igem.org/mediawiki/2018/a/a7/T--ASIJ_Tokyo--oggfp.pdf) + Our goal was to isolate A1AT secreted by all constructs so the protein can be accurately analyzed without obstructions. Two methods of purification, as well as several protein tags, were tested out. The two methods or column chromatography were His-tag specific protein purification with nickel column tubes and others were purified using the standard column chromatography method. Utilizing the His-tag was a major player in our success. We purified the proteins before conducting GFP fluorescence readings, the sialic acid assay (ELISA test), and gel electrophoresis. * b. [Sialic Acid Assay - original version of protocol](https://static.igem.org/mediawiki/2018/5/56/T--ASIJ_Tokyo--ogsialic.pdf) + The sialic acid content of a cell is a major characteristic differentiating mutated and unmutated variants. Therefore we chose to conduct a sialic acid assay as a means by which to attain quantitative results to determine with greater accuracy if the gene edit was successful. Based on previous literature, we determined that the unmutated constructs would secrete more sialic acid. The mutated constructs were expected to secrete less, due to its tendency to polymerize in the cell, preventing its ability to exit the cell. This assay would allow us to determine the sialic acid levels for each construct and thus allow us to compare the CRISPR edited construct with the unmutated construct and determine based on the proximity of the sialic acid contents if our gene edit was successful. If successful, the CRISPR edited constructs would show sialic acid secretion similar to that of the unmutated construct. Part VIII: Incorporation of New information to use His-tag * a. Redesign construct to include His-tag * b. Gibson assembly * c. Transformation * d. [Protein Purification using His Tag Protein Chromatography - redone Oct 10 (10 trials)](https://static.igem.org/mediawiki/2018/2/2b/T--ASIJ_Tokyo--histag.pdf) * e. [Sialic Acid Purification redone using new protocol (6 trials)](https://static.igem.org/mediawiki/2018/8/84/T--ASIJ_Tokyo--esla.pdf) * f. Fluoroscence Microplate Reading to Quantify Protein Concentration of Constructs using GFP standard curve + In another attempt to determine the optimal construct and determine whether the mutation was successfully corrected, we quantified the secretion of A1AT through a GFP reporter system. To create a standard curve showing GFP concentration and relative fluorescence intensity, we used concentrations of 0 uM, 8 uM, 16 uM, 32 uM, and 40 uM GFP in 1 mL PBS. Based on this curve, we took our fluorescence readings from our different constructs and used the equation Relative Fluorescence Intensity= - 0.015 + 0.007625(GFP Concentration), with R2 = 0.991927, to determine the concentration of GFP from the fluorescence intensity measured from each construct. * g. Fluorescence Microplate Reading to Quantify Sialic Acid Concentration using Sialic Acid Standard Curve + Since our previous sialic assay was unsuccessful due the extremely complex procedure and lack of conclusive results, we decided to conduct a commercial kit version. A sialic acid assay provides for a direct method to target the sialic acid secreted through a GFP reporter system. Our goal was to quantify the amount of sialic acid secreted by each construct. By doing so, we could compare the CRISPR edited construct with our unmutated construct to determine if our gene edit was successful. The mutated constructs would serve as a negative control, as its tendency to polymerize would result in the least sialic acid secretion. By using an antibody tagged with GFP, we were able to directly target the sialic acid secreted and thus quantify the fluorescence, which was directly correlated to the amount of sialic acid secreted. And, as expected, after 10 reading of each construct, we were able to find the average fluorescence intensity and thus determine the standard error for each, to conclude that our results were statistically significant. * h. [Final Polyacrylamide Gel Confirmation of Constructs](https://static.igem.org/mediawiki/2018/4/44/T--ASIJ_Tokyo--electro.pdf) + Because our original electrophoresis was not conclusive, due to a wrong concentration used in gel, we decided to take on a 12% SDS Page Electrophoresis. This would allow us to compare the CRISPR constructs with the unmutated constructs to determine if our gene edit was successful. We once again expect to see a mutated variant with a much greater mass than the unmutated variant and ideally a genetically modified variant similar to an unmutated variant.

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###### Icon used in the background on the home page made by [dDara](https://www.flaticon.com/authors/ddara) from <www.flaticon.com>

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# Team:ASIJ Tokyo/Results

* [HOME](https://2018.igem.org/Team%3AASIJ_Tokyo)
* [TEAM](https://2018.igem.org/Team%3AASIJ_Tokyo/Team)
  + [COLLABORATION](https://2018.igem.org/Team%3AASIJ_Tokyo/Collaborations)
  + [ATTRIBUTIONS](https://2018.igem.org/Team%3AASIJ_Tokyo/Attributions)
* [PROJECT](https://2018.igem.org/Team%3AASIJ_Tokyo/Description)
  + [CRISPR](https://2018.igem.org/Team%3AASIJ_Tokyo/CRISPR)
  + [DESIGN](https://2018.igem.org/Team%3AASIJ_Tokyo/Design)
  + [EXPERIMENT](https://2018.igem.org/Team%3AASIJ_Tokyo/Experiments)
  + [NOTEBOOK](https://2018.igem.org/Team%3AASIJ_Tokyo/Notebook)
  + [RESULTS](https://2018.igem.org/Team%3AASIJ_Tokyo/Results)
  + [DEMONSTRATE](https://2018.igem.org/Team%3AASIJ_Tokyo/Demonstrate)
* [PARTS](https://2018.igem.org/Team%3AASIJ_Tokyo/Parts)
  + [IMPROVE](https://2018.igem.org/Team%3AASIJ_Tokyo/Improve)
* [HUMAN PRACTICES](https://2018.igem.org/Team%3AASIJ_Tokyo/Human_Practices)
  + [OUR ACTIVITIES](https://2018.igem.org/Team%3AASIJ_Tokyo/Activities)
  + [Q&A](https://2018.igem.org/Team%3AASIJ_Tokyo/Questions)
  + [FUTURE](https://2018.igem.org/Team%3AASIJ_Tokyo/Future)* [SAFETY](https://2018.igem.org/Team%3AASIJ_Tokyo/Safety)

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# RESULTS

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## GFP Fluorescence

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![](https://static.igem.org/mediawiki/2018/a/af/T--ASIJ_Tokyo--gfpgraph.png)

    By quantifying the secretion of antitrypsin through a GFP reporter system, we determined our optimal construct design. To create a standard curve showing GFP concentration and relative fluorescence intensity, we used concentrations of  **0 uM, 8 uM, 16 uM, 32 uM**, and **40 uM** GFP in 1 mL PBS. Based on this curve, we took our fluorescence readings from our different constructs and used the equation

Relative Fluorescence Intensity=-0.015 + 0.007625(GFP Concentration), with R2 = 0.991927

 to determine the concentration of GFP from the fluorescence intensity measured from each construct. The unmutated construct with the his and osmY tag and a single terminator system had the highest relative mean fluorescence of **0.1616** and the highest GFP concentration of **23.1607 uM**. To further prove that the construct with the his and osmY tag was the most effective, the data shows that the CRISPR gene edited construct had a relative mean fluorescence intensity of **0.1221** and a GFP concentration of **17.9803 uM**, which is higher than the unmutated constructs with double osmY tags with single and double terminator systems, which only had relative fluorescence intensities of **0.0423** and **0.0383** and GFP concentrations of **7.51475 uM** and **6.99016 uM**, respectively. Thus, looking into future projects using unmutated and CRISPR edited sequences of SERPINA1, a construct designed with a his and osmY secretion tag with a single terminator system will promise optimal secretion, as indicated in these fluorescent readings.

## Sialic Acid

---

![](https://static.igem.org/mediawiki/2018/8/8d/T--ASIJ_Tokyo--sialicacidgraph.png)

    The efficacy of our constructs were also quantified through an ELISA, in which we linked a fluorescence antibody to sialic acid, to directly measure the amount secreted by each construct. Sialic acid linked to GFP with concentrations of **0 uM, 10 uM, 20 uM, 30 uM, 40 uM, 60 uM, 80 uM**, and **100 uM** were used to create a standard curve with equation

 Relative Fluorescence Intensity= -0.006725 + 0.060599(Sialic Acid Concentration), with R2 = 0.998676

to determine the concentration of sialic acid from the fluorescence intensity measured from each construct. The CRISPR construct with double osmY secretion tags and double terminators had 100% of the sialic acid levels of the unmutated construct, indicating its efficacy in correcting the point mutation, and secreting expected sialic acid levels. However, its overall low relative fluorescence intensity at **0.620211667** and concentration of **10.3456667 uM** reveals the double osmY and double terminator constructs to be ineffective, and thus may not promise optimal results in future experiments, regardless of the success of the CRISPR gene edit in this construct. The constructs with the double osmY secretion tag and a single terminator showed the CRISPR edited construct to secrete **75.4376%** of the amount of sialic acid secreted by the unmutated variant, showing success of the CRISPR gene edit. Similarly, the constructs with the his and osmY tag with a single terminator showed the CRISPR edited construct to secrete **74.934689%** of the amount of sialic acid secreted by the unmutated variant, showing success of the CRISPR gene edit as well. The relative sialic acid concentration secreted by the unmutated and CRISPR gene edited constructs with the his and osmY tag with a single terminator was **80.9585 uM** and **60.666 uM** respectively, whereas the unmutated and CRISPR gene edited constructs with a double osmY tag and a single terminator showed sialic acid concentrations of **51.074 uM** and **38.529 uM**, respectively. Because the percentage of sialic acid secreted by the construct with the his and osmY tag with a single terminator, **74.934689%**, and by the construct with the double osmY tag and a single terminator, **75.4376%**, was similar, we can use the sialic acid concentrations in determining which construct would demonstrate overall efficacy. In doing so, we can conclude that the construct with the his and osmY tag with a single terminator maximizes sialic acid secretion of both the unmutated and CRISPR edited construct. In addition, we can confirm the polymerization of the protein in the mutated constructs, as sialic acid levels of the mutated construct as a percentage of the unmutated construct remained low at **2.9548545%, 3.3843051%**, and **9.7961919%** for the double osmY tag and double terminator system, double osmY tag and single terminator system, and his and osmY tag and single terminator system, respectively.

## Electrophoresis

---

![](https://static.igem.org/mediawiki/2018/0/0e/T--ASIJ_Tokyo--%2AResults_Electrophoresis%2A.jpg)

1. Mutated SERPINA1 with double osmY and double terminator
2. Unmutated SERPINA1 with double osmY and double terminator
3. CRISPR gene edited SERPINA1 with double osmY and double terminator
4. Mutated SERPINA1 with double osmY and single terminator
5. Unmutated SERPINA1 with double osmY and single terminator
6. CRISPR gene edited SERPINA1 with double osmY and single terminator
7. Mutated SERPINA1 with his and osmY tags and single terminator
8. Unmutated SERPINA1 with his and osmY tags and single terminator
9. CRISPR gene edited SERPINA1 with his and osmY tags and single terminator

    In the page electrophoresis stainings, we can confirm that the mutated SERPINA1 constructs in wells 1, 4, and 7 with denser bands around 40 kDa indicate its tendency to polymerize. The unmutated SERPINA1 constructs in wells show bands ranging from **30 kDa** to **60 kDa**. The CRISPR edited SERPINA1 in wells **3**, **6**, and **9** show the similar dense bands expressed in the mutant variants, of **40 kDa**, however also do indicate small levels of protein at weights of **60 kDa**, **50 kDa**, and **30 kDa**, which are also observed in the unmutated variants of SERPINA1. Because the CRISPR constructs show hybrid characteristics of both the bands from the unmutated and mutated constructs, we may have created an MZ mutant variant. Regardless, because the gel electrophoresis indicated the unmutated, mutated, and CRISPR edited variants to separate differently in the gel, the results remain conclusive and consistent with our other experiments.

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###### Icon used in the background on the home page made by [dDara](https://www.flaticon.com/authors/ddara) from <www.flaticon.com>