iGEM 2021 — Team Gifu

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Gifu/Notebook

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Gifu/Engineering

[iGEM Gifu](https://2021.igem.org/Team%3AGifu)

* [Home](https://2021.igem.org/Team%3AGifu)
* Project

  [Description](https://2021.igem.org/Team%3AGifu/Description)
  [Background](https://2021.igem.org/Team%3AGifu/Design)
  [Safety](https://2021.igem.org/Team%3AGifu/Safety)
  [Contribution](https://2021.igem.org/Team%3AGifu/Contribution)
  [Implementation](https://2021.igem.org/Team%3AGifu/Implementation)
  [Parts](https://2021.igem.org/Team%3AGifu/Parts)
  [Improve](https://2021.igem.org/Team%3AGifu/Improve)
* Experiment

  [Result](https://2021.igem.org/Team%3AGifu/Result)
  [Modeling](https://2021.igem.org/Team%3AGifu/Model)
  [Engineering](https://2021.igem.org/Team%3AGifu/Engineering)
* Human Practice

  [Human Practice](https://2021.igem.org/Team%3AGifu/Human_Practices)
  [Education & Communication](https://2021.igem.org/Team%3AGifu/Communication)
  [Arts](https://2021.igem.org/Team%3AGifu/Another_Area)
* Team

  [Member](https://2021.igem.org/Team%3AGifu/Member)
  [Attribution](https://2021.igem.org/Team%3AGifu/Attributions)
  [Collaboration](https://2021.igem.org/Team%3AGifu/Collaborations)
* [Judging](https://2021.igem.org/Team%3AGifu/Judging)

# Engineering

## Engineering success

# **Introduction**

Our laboratory activities were greatly restricted by the COVID-19 outbreak. In the following figure, the area surrounded by a red frame is the period when activities in the university were prohibited due to the emergency declaration issued by the Japanese government, measures to prevent the spread of the infection, and university regulations, and the yellow area is the period when we refrained from activities in the lab since the infection was spreading in Japan. We had a very limited period of time to conduct our experiments, which lasted a month and a half in July and the second half of October. Therefore, we discussed our project with many experts through the [Human Practice](https://2021.igem.org/Team%3AGifu/Human_Practices) to improve the project and design it to be used after the success of the project. At the beginning of the project, we were thinking of using Aptamer for detection, but after exploring simple quantification methods using microfluidic plates and Toehold switch RNA, we finally decided to use Cas12a, which has been shown to be useful for quantification of nucleic acids in previous studies, to quantify HHV-6. We decided to quantify HHV-6 using Cas12a, which has been shown to be useful for nucleic acid quantification in previous studies. After deciding on this project, we had repeated discussions with experts to confirm the improvements and the usefulness of the design for social implementation.Through these activities, we repeated the cycle of design, build, test, and improving our projects. This was also the case in the wet lab. In the limited time we had, we experimented, revised, and tested each time.

![](https://static.igem.org/mediawiki/2021/3/3c/T--Gifu--covid-19.jpg)

Although the experiments were conducted in a very short period of time, during July and the second half of October, we proceeded with our lab experiments and worked for the success of our project. We needed to express Cas12a and synthesize crRNA to make our project a success. We designed an experiment to demonstrate the DNA recognition ability and cleavage activity of Cas12a on artificially synthesized U94, and then to confirm the collateral cleavage activity of our Cas12a using T7phage after the expression of Cas12a and synthesis of crRNA. The design of the experiment is as follows.

## Cycle1. Coxstruction of Cas12a ~*iVEC*~

# **Design**

The parts were designed as the following DNA circuit.

![](https://static.igem.org/mediawiki/2021/8/88/T--Gifu--engineering1.png)

Figure1. DNA circle of Lbcpf1 (Cas12a)

As a coding sequence (CDS), we designed Lbcpf1 (Cas12a) fused with a 10× histidine tag and MBP tag with a TEV site in between. The TEV site was added after Cas12a purification to separate the tag from Cas12a. The TEV protease recognizes the TEV site (lu-Asn-Leu-Tyr-Phe-Gln-Gly/Ser) and cleaves between Gln and Gly (or Ser) with high specificity. We designed a composite part of the CDS by adding a T7promoter and RBS at the 5' end and a double terminator at the 3' end.

# **Build**

Our parts are about 5400 bp, which is over the 1800 bp limit of Twist's DNA synthesis service. Therefore, we had to order this sequence in three separate fragments and assemble these. Both ends of each fragment were designed to be 40 bp complementary, and we decided to add overhang sequences to the 5' end of fragment 1 and the 3' end of fragment 3 by PCR to join them to the backbone. We then tried to clone the amplicons of each fragment by *i*VEC.

# **Test**

We amplified each DNA fragment by PCR using the KAPA hifi hot start mix (KAPA biosystems). Click the button for protocols.

[Click Here for Protocols](https://www.protocols.io/view/amplification-of-dna-bzbcp2iw)

We then tried to clone them by *iVEC*, but the cloning was not successful. Click the button for protocols.

[Click Here for Protocols](https://www.protocols.io/view/in-vivo-cloning-ivec-bzcmp2u6)

![](https://static.igem.org/mediawiki/2021/3/3e/T--Gifu--ivec.jpg)

Figure2. DNA circle of Lbcpf1 (Cas12a)

No target colonies were found in all of the negative control, condition A, and condition B, confirming that the GFP-expressing pSB1C3 used as the Backbone template was incorporated.

# **Learn**

The reason for the failure of the *in vivo* assembly was that the transgene fragment was too long, resulting in poor ligation and a significant decrease in transformation efficiency. Therefore, we constructed the constructs by performing *in vitro* Gibson assembly before transformation. We also modified the PCR protocol for Backbone linearization to prevent the incorporation of Backbone's Template DNA, and decided to perform PCR with a reduced amount of DNA template.

## Cycle2. Construction ofCas12a ~in vitro assembly~

# **Design**

Based on the results of Cycle 1, we decided to transform NEB-10beta after combining each fragment with Gibson assembly.

# **Build**

For in vitro assembly of DNA fragments, we decided to use NEBuilder®, an assembly kit based on Gibson Assembly, which first degrades the ends of DNA fragments by exonuclease. In NEBuilder®, the ends of the DNA fragments are first degraded by exonucleases, which hybridize the sticky ends, followed by DNA polymerase, which synthesizes the degraded strand. Finally, DNA ligase repairs the nick and completes the DNA assembly. The NEB® 10-beta competent cell was used for the subsequent transformation, which is expected to solve the problem of in vivo cloning, since the cloning efficiency of the NEB® 10-beta is slightly reduced even with plasmids of high molecular weight. In addition, the backbone was changed from pSB1C3 to pSB1A3. pSB1C3 as the backbone uses chloramphenicol for screening. Chloramphenicol has the potential to inhibit protein synthesis and thus inhibit the expression of Cas12a. We changed the backbone to pSB1A3 for screening with ampicillin.

# **Test**

**1. fragment and backbone amplification by PCR**
Click the button for protocols.

[Click Here for Protocols](https://www.protocols.io/view/amplification-of-dna-bzbcp2iw)

**2. Gibson assembly**
Click the button for protocols.

[Click Here for Protocols](https://www.protocols.io/view/in-vitro-assembly-and-transformation-bzc9p2z6)

**3.Direct colony PCR**
PCR on a fully constructed plasmid with Fw primers for fragment 1 and Rv primers for fragment 3 yields an insert sequence of 5400 bp. We confirmed the constructs by applying direct colony PCR to a single stand colony that we grew by transformation.
**4. Sanger sequencing**
We performed Sanger sequencing on the plasmid in which the 5400 bp insert sequence was confirmed, to check the junction part of each fragment.

![](https://static.igem.org/mediawiki/2021/a/a3/T--Gifu--result4.png)

Figure3. Overview of Sanger sequencing

From the results of the Sanger sequence, we confirmed that the full construct was successful.

# **Learn**

In this cycle, we were able to assemble the three fragments encoding Cas12a together with the backbone to obtain a full construct. We then moved on to the next step of expressing Cas12a from this plasmid.

## Cycle3. Expression

# **Design**

We transformed the constructed plasmid into BL21 (DE3) and tried to express Cas12a.

# **Build**

We ran the 2018 Doudna's method and The University of Queensland's culture method and optimized the culture method to get more Cas12a.

# **Test**

**1. Transformation into BL21**
Place the frozen competent cell on ice to melt. 2. Add 1 to 2 µl of plasmid to the competent cell and mix by gentle pipetting. Add 1 ml of Soc medium (room temperature), mix by inverting, and incubate in a water bath incubator at 37°C for 45 minutes. 6. After incubation, centrifuge the cells at 5,000 g for 1 minute at room temperature, discard about 100 µl of the supernatant, and wash the precipitated cells with the remaining supernatant.
6. centrifuge at 5,000 g for 1 minute at room temperature, discard about 100 µl of the supernatant, and stain the precipitated cells with the remaining supernatant.
Spread the entire volume on agar medium supplemented with 100 mg/ml ampicillin at x500, and incubate overnight at 37°C.
**2. Expression of Cas12a**
TEV treatment and Ni-NTA purification
After the cells were disrupted by sonication, we purified them by Ni-NTA column. The protein we designed is fused with 10 consecutive histidine tags (x10 His Tag) and MBP tag, so it can be trapped and purified by Ni-NTA column. The TEV sequence located between the tag and Cas12a was then cleaved by TEV protease to separate the tag and Cas12a. This solution was purified again by Ni-NTA column to trap the tag in the column and obtain pure Cas protein. The TEV protease that we used for tag cleavage has a His tag attached to it, which can be recovered with the tag cleaved by the Ni-NTA column. This purified protein was confirmed by SDS. Click the button for protocols.

[Click Here for Protocols](https://www.protocols.io/view/protein-purification-bzcsp2we)

**3. SDS-PAGE gel**
The molecular weight of Cas12a is 143 kDa, and the protein containing the tag is approximately 180 kDa. As a result of PAGE, we could not confirm the expression of Cas12a.

[Click Here for Protocols](https://www.protocols.io/view/sds-page-bzdqp25w)
![](https://static.igem.org/mediawiki/2021/b/b5/T--Gifu--sds.jpg)

Figure4. SDS-PAGE gel of Crude extraction

# **Learn**

Normally, the addition of IPTG would induce protein expression under the T7 promoter, and a large amount of protein would be obtained. However, we were unable to obtain a sufficient amount of Cas12a with our culture method. We thought that there might have been a problem with the culture method, so we followed the Cas9/Cas12a expression protocol published by The University of Queensland and induced expression again. In this method, the incubation time, the scale of incubation, and the concentration of IPTG to be added are significantly different from the original protocol. However, we could not confirm the expression of Cas12a. At the same time, we transformed part BBa\_K199118 in the iGEM kit into BL21 (DE3) and cultured it, in the same way, using three different media, Terrific broth, 2YT broth, and LB broth, and compared the expression levels. This part encodes RFP, and if RFP is expressed, it can be confirmed by the naked eye.
The results of this experiment showed that the expression levels in the Terrific broth, 2YT broth, and LB broth were so different that the differences were visible to the naked eye. From this result, we confirmed that there is no problem with Doudna's Cas12a protocol using Terrific broth.

![](https://static.igem.org/mediawiki/2021/4/4f/T--Gifu--figure5.png)

Figure5. Expression of RFP from each culture medium (From left to right: Terrific broth, 2YT broth, LB broth)

## Cycle4. Expression & Purification

# **Design**

From the results above, it was confirmed that there was no problem with the culture method, so we tried to obtain pure Cas12a by culturing again and purifying by Ni-NTA column and TEV treatment.

# **Build**

At this stage, TEV treatment and purification with Ni-NTA column were performed for the first time, and since the reaction time was 1-4 hours according to the TEV treatment protocol, we tried to optimize the TEV treatment protocol by performing TEV treatment at 1, 2, and 4 hours respectively and comparing the yields.

# **Test**

**1. Transformation into BL21**
The transformation was carried out by the following protocol.
**2. Expression of Cas12a**
Cas12a according to the Cas12a expression protocol described in Doudna's paper in 2018.
**3. TEV treatment and Ni-NTA purification**
After disruption of the cells by sonication, we purified the protein by Ni-NTA column. The protein we designed contains 10 consecutive histidine tags (×10 His Tag) and MBP tags fused together, which can be trapped and purified by Ni-NTA column. The TEV sequence located between the tag and Cas12a was then cleaved by TEV protease to separate the tag and Cas12a. This solution was purified again by the Ni-NTA column to trap the tag in the column and obtain pure Cas protein. The TEV protease that we used for tag cleavage has a His-tag attached to it, which can be recovered with the tag cleaved by the Ni-NTA column. This purified protein was confirmed by SDS.
**4. SDS-PAGE gel**
The molecular weight of Cas12a is 143 kDa, and the protein containing the tag is approximately 180 kDa. As a result of PAGE, we could not confirm the expression of Cas12a.
**5. Measuring the concentration of protein solutions**
The concentrations of the TEV-treated and purified protein solutions were measured using TOMY's Micro-volume-Spectrophotometer. 0.023 mg/ml for 1 hour of TEV treatment, 0.048 mg/ml for 2 hours, and 0.054 mg/ml for 4 hours were observed. The difference in concentration between 1 hour and 2 hours was large, while the difference between 2 hours and 4 hours was small. From these results, it can be inferred that the maximum yield of TEV treatment can be obtained in approximately 4 hours.
![](https://static.igem.org/mediawiki/2021/2/2a/T--Gifu--result5.png)

Figure6. SDS-PAGE gel of Cas12a

# **Learn**

The results of Cycle 3 and Cycle 4 showed that high expression of the target protein by Terific broth was possible and that high expression of Cas12a could be expected by this method. However, Cycle4 did not show Cas12a expression; if Cas12a had been degraded, we would have seen an unclear band in a large area of the lane.If Cas12a was degraded, we would have seen unclear bands over a large area of the lane, but we did not find any such bands in our experiment. These results suggest that Cas12a may not be expressed in the cells. We considerd a new design for the case where Cas12a is not expressed, and summarized it in the following Next design.

## Cycle5. crRNA synthesis

# **Design**

The DNA encoding crRNA was designed as shown in the figure above. crRNA consists of a consensus sequence necessary for complex formation with Cas12a and a sequence that binds to the target. By adding the T7 promoter to the 5' end of the designed crRNA coding DNA, RNA can be synthesized by in vitro transcription using the T7 polymerase.

![](https://static.igem.org/mediawiki/2021/0/0b/T--Gifu--cycle5.png)

Figure7. crRNA

# **Build**

To test the cleavage activity by Cas12a, we synthesized crRNA that forms a complex with Cas12a.

# **Test**

**1. PCR of gRNA**
**2. Ethanol precipitation**
One-tenth volume of 3M sodium acetate and 2.5 times volume of ethanol were added to the DNA solution and mixed. The tube was incubated at -20°C without over, and then centrifuged at 15,000 rpm for 20 minutes to remove the supernatant. Then 70% ethanol was added and the tubes were centrifuged at 15,000 rpm for 5 minutes, and the supernatant was removed again and dried.
**3. Transcription and purification**
The transcription was done according to the protocol of the T7 MEGAscript kit, and the reaction was done overnight to obtain a higher yield. RNA was then purified by phenol:chloroform extraction and isopropanol precipitation according to the method described in this protocol.
**4. Bioanalyzer**
RNA nano chips were used for the bioanalyzer analysis, and all operations were performed according to the Agilent RNA6000 nano chip kit guide.

![](https://static.igem.org/mediawiki/2021/e/e3/T--Gifu--result6.png)

Figure8. Qualitative and quantitative testing of crRNA by bioanalyzer

As a result of the bioanalyzer, we were able to confirm the presence of the target sequence in one of the crRNAs for T7 phage and two of the crRNAs for HHV-6. However, the amount of confirmed crRNA was very small.

# **Learn**

We need to revise our transcription method. For example, this time we did not include the terminator sequence in the crRNA coding DNA. This may have reduced the transcription efficiency.
T7 MEGA We tried to optimize the transcription by extending the reaction time and expanding the reaction system according to the method described in the script kit protocol, but it was not enough. We need to investigate the transcription efficiency of short DNA with and without terminator sequences in future work.

## Cycle6. DNA cleavage assay

# **Design**

We performed DNA cleavage assay using Cas12a obtained in Cycle 3 and crRNA obtained in Cycle 5 to validate our system.

# **Build**

DNA cleavage assay was performed to test the on-target effect of Cas12a. Cas12a forms a complex with crRNA and then recognizes and cleaves dsDNA complementary to the crRNA. Prior to the quantification of DNA using Cas12a, we tested whether Cas12a has an on-target effect, which is the beginning of its function.

# **Test**

**1. Amplification of U94**
HHV-6-derived ORF94 (U94) used for validation was amplified by PCR.
**2. Purification of U94**
The U94 amplicon was purified using a Gel/PCR extraction kit (Nippon genetics).
**3. DNA cleavage assay**
DNA cleavage assay was performed according to Doudna's paper in 2018. Each mixture was incubated at 37°C overnight.We then checked for cleavage by agarose gel electrophoresis, but no DNA cleavage was observed. Click the button for protocols.

[Click Here for Protocols](https://www.protocols.io/view/cleavage-assay-bzdmp246)

![](https://static.igem.org/mediawiki/2021/1/17/T--Gifu--result2.png)

Figure9. agarose gel electrophoresis of a Cleavage assay

# **Learn**

Based on the results of the DNA cleavage assay, we suspected that Cas12a might be losing its activity, and sought a protocol for the next experiment.
We did not add the reducing agent tris (2-carboxyethyl) phosphine (TCEP) at the BL21 disruption step, as TCEP has strong reducing properties and high stability, and is added to the reaction to maintain the activity of the protein by preventing disulfide bonds between proteins. We decided that we needed to try a protocol where this reagent was added to the reaction.

## Next design

In order to make Cas12a more tractable, we performed a silent mutation of the Cas12a sequence to remove the type IIS recognition site. This mutation was done according to the codon usage of E. coli and was performed in such a way as to avoid large-scale mutations such as frameshifting. However, it is possible that the mutation occurred during these manipulations or during PCR amplification. In order to confirm the mutation, we need to read the entire sequence of the assembled full construct. This will allow us to confirm whether the mutation caused the loss of activity.

Doudna's design for expression was used for most of the DNA parts designed in this experiment, but the T7 terminator was designed by replacing it with a double terminator (BBa\_B0014) because it could not be synthesized by Twist's DNA synthesis service due to GC ratio issues. In order to confirm whether the correct RNA encoding Cas12a is expressed, we extracted RNA from BL21 culture medium and analyzed it by bioanalyzer. This can be confirmed by extracting RNA from the BL21 culture medium and analyzing it with a bioanalyzer. If this experiment does not show the 3684 nt of RNA that is thought to encode Cas12a, we will need to redesign the terminator and promoter.

We searched (TTTN), the PAM sequence of Cas12a, against T7p47 of T7 Phage and ORFU94 of HHV-6 to obtain the downstream sequences. We selected sequences with high GC rates and then performed homology searches on these sequences using nBLAST, because we thought that sequences with high GC rates would have higher specificity for binding. This series of design steps eliminated sequences with low concordance to the human genome and to bacteria thought to be present in the human oral cavity, and which were likely to act as off-targets for Cas12a. In this study, three crRNAs were selected from HHV-6 and T7phage, respectively, and used in the experiments. However, there are many candidate sequences for crRNAs, and it is necessary to perform DNA cleavage assay using these candidates to find the best crRNA. We selected other candidates as our next design and summarized them in the table below. We believe that running the engineering cycle again on these candidates will lead to the success of the project.

Table1. crRNA for T7 Phage(left) and crRNA for HHV-6(right)

![](https://static.igem.org/mediawiki/2021/f/fb/T--Gifu--table1.png)

## Future work ~validation of our system~

Assuming that Cas12a and crRNA were correctly obtained in the engineering cycle described above, we considered validation by cleavage activity test.
The validation is divided into the following three stages.

**1. on-target activity test using amplicon of U94**
**2. Optimization of DNA quantification test using amplicon of T7phage-derived standard sequence**
**3. DNA quantification test using the T7phage genome**

The first step of validation is DNA recognition and on-target activity test using U94 amplicon. If the expressed Cas12a functions properly, the following figure is expected to be obtained.
![](https://static.igem.org/mediawiki/2021/0/0e/T--Gifu--ladder.jpg)

Figure10. in silico simulation of on-target activity test of Cas12a with U94 amplicon

Next, a DNA quantification test is performed using amplicons of the T7 Phage-derived standard sequence. In this experiment, the standard sequence and fluorescent probe are added to the pre-assembled Cas12a-crRNA complex. The test is performed by diluting the target concentration in steps, and a calibration curve is constructed from the fluorescence intensity. This allows us to confirm whether Cas12a has collateral activity and whether there is a correlation between the amount of DNA and the fluorescence intensity. In this experiment, we will also optimize the variables in our DNA quantification system, such as the concentration of crRNA and the concentration of Cas12a. If this experiment is successful, we expect to obtain results similar to what we have been modeling.

Finally, the T7 Phage genome will be tested using DNA extracted by spin column method after the T7 Phage step dilution, and the fluorescence intensity will be measured to create a calibration curve. This test confirms whether a reaction is occurring with the DNA extracted from the virus and whether there is a correlation between sample concentration and fluorescence intensity. This will show that our system is functioning properly. Also, as mentioned in the [Implementation](https://2021.igem.org/Team%3AGifu/Implementation) section, our goal is to eventually create a system that can measure fluorescence using a device that many people have, such as a smartphone. In order to optimize and complete this system, we need to validate the measurement with a smartphone. Check our [Implementation](https://2021.igem.org/Team%3AGifu/Implementation) for more information.

## References

1)Chen JS, Ma E, Harrington LB, Da Costa M, Tian X, Palefsky JM, Doudna JA. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science. 2018 Apr 27;360(6387):436-439. doi: 10.1126/science.aar6245. Epub 2018 Feb 15. Erratum in: Science. 2021 Feb 19;371(6531): PMID: 29449511; PMCID: PMC6628903.
2) The University of Queensland. CRISPR-Cas9/Cas12A expression, purification and validation.https://gih.uq.edu.au/research/gene-editing/crispr-cas9cas12a-expression-purification-and-validation
3)Sharp PM, Li WH. The codon Adaptation Index--a measure of directional synonymous codon usage bias, and its potential applications. Nucleic Acids Res. 1987 Feb 11;15(3):1281-95. doi: 10.1093/nar/15.3.1281. PMID: 3547335; PMCID: PMC340524.

**Sponsors**
[IDT](https://sg.idtdna.com/pages)
[CosmoBio](https://www.cosmobio.co.jp)
[Twist biosciences](https://twistbioscience.com)
[SnapGene](https://www.snapgene.com)
[New England Biolabs](https://international.neb.com)
[Promega](https://www.promega.jp/)
[CCi CORPORATION](https://cci-corporation.com/)
[KINJIRUSHI Co.,Ltd.](https://www.kinjirushi.co.jp/)

**Information**
[Twitter](https://twitter.com/iGEMgifu)
Contact
[Gifu University](https://www.gifu-u.ac.jp/en/)
[Home](https://2021.igem.org/Team%3AGifu)

iGEM GIFU is a research group of Gifu University,
based in Gifu, Gifu prefecture Japan

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# Team:Gifu/Experiments

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Gifu/Results

# Results

You can describe the results of your project and your future plans here.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)