iGEM 2021 — Team Kyoto

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# Team:Kyoto/Notebook

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* [Description](https://2021.igem.org/Team%3AKyoto/Description)
* [Implementation](https://2021.igem.org/Team%3AKyoto/Implementation)
* [Proof of Concept](https://2021.igem.org/Team%3AKyoto/Proof_Of_Concept)
* [Engineering](https://2021.igem.org/Team%3AKyoto/Engineering)
* Dry Lab
  + [Modeling](https://2021.igem.org/Team%3AKyoto/Model)
  + [Software](https://2021.igem.org/Team%3AKyoto/Software)
  + [Hardware](https://2021.igem.org/Team%3AKyoto/Hardware)
* Wet Lab
  + [Experiments](https://2021.igem.org/Team%3AKyoto/Experiments)
  + [Results](https://2021.igem.org/Team%3AKyoto/Results)
  + [Parts](https://2021.igem.org/Team%3AKyoto/Parts)
  + [Notebook](https://2021.igem.org/Team%3AKyoto/Notebook)
  + [Safety and Security](https://2021.igem.org/Team%3AKyoto/Safety)
* People
  + [Human Practices](https://2021.igem.org/Team%3AKyoto/Human_Practices)
  + [Education and Communication](https://2021.igem.org/Team%3AKyoto/Communication)
  + [Sustainable Development
    Impact](https://2021.igem.org/Team%3AKyoto/Sustainable)
  + [Excellence in Another Area](https://2021.igem.org/Team%3AKyoto/Gift_from_Synthetic_Biology)
  + [Contribution](https://2021.igem.org/Team%3AKyoto/Contribution)
* Teams
  + [Team](https://2021.igem.org/Team%3AKyoto/Team)
  + [Collaborations](https://2021.igem.org/Team%3AKyoto/Collaborations)
  + [Partnership](https://2021.igem.org/Team%3AKyoto/Partnership)
  + [Attributions](https://2021.igem.org/Team%3AKyoto/Attributions)
* Awards
  + [Result](https://2021.igem.org/Team%3AKyoto/Awards)
  + [Criteria](https://2021.igem.org/Team%3AKyoto/Criteria)

Notebook

| Number | Title |
| --- | --- |
| 1 | [Yuto Ueda's note](https://static.igem.org/mediawiki/2021/b/b0/T--Kyoto--01_Labnote.pdf) |
| 2 | [Nao Tajima's note](https://static.igem.org/mediawiki/2021/f/fb/T--Kyoto--02_Labnote.pdf) |
| 3 | [Kodai Shiotsu & Ryusei Hayamatsu's note](https://static.igem.org/mediawiki/2021/2/2f/T--Kyoto--03_Labnote.pdf) |
| 4 | [Yuki Kobayashi's note](https://static.igem.org/mediawiki/2021/d/dd/T--Kyoto--04_Labnote.pdf) |
| 5 | [Hiroto Koga's note](https://static.igem.org/mediawiki/2021/1/13/T--Kyoto--05_Labnote.pdf) |
| 6 | [Chihiro Goya & Nao Tajima's note](https://static.igem.org/mediawiki/2021/5/5f/T--Kyoto--06_Labnote.pdf) |
| 7 | [Chisato Nishizawa's note](https://static.igem.org/mediawiki/2021/c/c8/T--Kyoto--07_Labnote.pdf) |
| 8 | [Chihiro Goya's note](https://static.igem.org/mediawiki/2021/b/b6/T--Kyoto--08_Labnote.pdf) |
| 9 | [Alexander Liu's note](https://static.igem.org/mediawiki/2021/e/e9/T--Kyoto--09_Labnote.pdf) |

---

Contact Us

Instagram: @igemkyoto

Twitter: @iGEMkyoto

E-mail: igemkyoto2021[at]gmail.com

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# Team:Kyoto/Engineering

[![](https://static.igem.org/mediawiki/2021/2/22/T--Kyoto--images--logoside1.png)](https://2021.igem.org/Team%3AKyoto)

* [Description](https://2021.igem.org/Team%3AKyoto/Description)
* [Implementation](https://2021.igem.org/Team%3AKyoto/Implementation)
* [Proof of Concept](https://2021.igem.org/Team%3AKyoto/Proof_Of_Concept)
* [Engineering](https://2021.igem.org/Team%3AKyoto/Engineering)
* Dry Lab
  + [Modeling](https://2021.igem.org/Team%3AKyoto/Model)
  + [Software](https://2021.igem.org/Team%3AKyoto/Software)
  + [Hardware](https://2021.igem.org/Team%3AKyoto/Hardware)
* Wet Lab
  + [Experiments](https://2021.igem.org/Team%3AKyoto/Experiments)
  + [Results](https://2021.igem.org/Team%3AKyoto/Results)
  + [Parts](https://2021.igem.org/Team%3AKyoto/Parts)
  + [Notebook](https://2021.igem.org/Team%3AKyoto/Notebook)
  + [Safety and Security](https://2021.igem.org/Team%3AKyoto/Safety)
* People
  + [Human Practices](https://2021.igem.org/Team%3AKyoto/Human_Practices)
  + [Education and Communication](https://2021.igem.org/Team%3AKyoto/Communication)
  + [Sustainable Development
    Impact](https://2021.igem.org/Team%3AKyoto/Sustainable)
  + [Excellence in Another Area](https://2021.igem.org/Team%3AKyoto/Gift_from_Synthetic_Biology)
  + [Contribution](https://2021.igem.org/Team%3AKyoto/Contribution)
* Teams
  + [Team](https://2021.igem.org/Team%3AKyoto/Team)
  + [Collaborations](https://2021.igem.org/Team%3AKyoto/Collaborations)
  + [Partnership](https://2021.igem.org/Team%3AKyoto/Partnership)
  + [Attributions](https://2021.igem.org/Team%3AKyoto/Attributions)
* Awards
  + [Result](https://2021.igem.org/Team%3AKyoto/Awards)
  + [Criteria](https://2021.igem.org/Team%3AKyoto/Criteria)

Engineering Success

The following is a record of our Engineering Flow.

We have set a major goal to make the plant production more sustainable.
To achieve this goal, we established the four measures.
For each topic, we proceeded with the project in accordance with the Principles of Engineering.

Click each title to see detail

1. Virus and Viroid detection

2. Feeding RNAi for western flower thrips

3. Other applications for cut flower maintenance

4. Biomolecular Production Platform

- Click to visit -

* [Virus and Viroid detection](#sctarget0)
* [Cycle1](#sctarget1)
* [Cycle2](#sctarget2)
* [Cycle3](#sctarget3)
* [Cycle4](#sctarget4)
* [Cycle5](#sctarget5)
* [Cycle6](#sctarget6)
* [References](#sctarget7)

Virus and Viroid detection

*[Cycle1
We performed CRISPR-Cas12a mediated LAMP assay
to detect DNA viruses including Dahlia Mosaic Virus (DMV) using commercially available enzymes](#sctarget1)
[Cycle2
We performed CRISPR-Cas12a mediated
RT-LAMP assay to detect RNA viruses such as CSVd or TSWV using purchased enzymes](#sctarget2)
[Cycle3
We reduced the cost of one reaction by
producing homemade enzymes](#sctarget3)
[Cycle4
We created hardware which helps farmers
to do point-of-care testing](#sctarget4)
[Cycle5
We created image recognition software to
improve the performance of the CRISPR-Cas12a-mediated LAMP/RT-LAMP assay](#sctarget5)
[Cycle6
We designed a portable detection package
and improved a cycle of diagnostic imaging accuracy](#sctarget6)*

Cycle1
  We performed
CRISPR-Cas12a mediated LAMP assay to detect DNA viruses including Dahlia Mosaic
Virus (DMV) using commercially available enzymes

Design

We first tried to detect Dahlia Mosaic Virus (DMV) which has become a serious problem for the Japanese
flower industry. When dahlias are infected with the virus, their leaves show weird patterns, losing
their commercial value and ending up being discarded. When we interviewed several experts during human
practice, we learned that RT-qPCR is now commonly used by them to detect the virus in dahlia bulbs.
However, since the RT-qPCR method requires expensive and complicated equipment and knowledge of
molecular biology, farmers are not able to detect virus infections by themselves. To solve this
problem, we worked to create a new detection method for DMV that is easy to handle[1.1].Therefore, we
focused on the LAMP method (Loop-Mediated Isothermal Amplification)[1.2].
The LAMP method amplifies
DNAs
simply by incubating reactions at isothermal temperatures. Since it is easier and faster than PCR, it
is frequently used for virus detection.[1.3][1.4]

![](https://static.igem.org/mediawiki/2021/6/6c/T--Kyoto--Engineering_050.png)

*How does the LAMP method work?*
In the LAMP method, a strand-displacing B. stearothermophilus DNA polymerase initiates DNA synthesis
from primers attached to the end of the target sequence. The ssDNA amplicon forms a loop at the end of
it, which works as a new template for further DNA amplification. Thus, DNA is amplified
exponentially.[1.5]

![](https://static.igem.org/mediawiki/2021/7/7e/T--Kyoto--Engineering_LAMP_sashikae.png)

However, one of the disadvantages of this LAMP method is that it is likely to produce many
pseudo-positive samples due to non-specific amplification. This is a fatal problem because the
unnecessary disposal of pseudo-positive lines also leads to economic losses for the flower industry.
To compensate for this weakness of the LAMP method, we decided to combine the LAMP method and
CRISPR-Cas12a fluorescence assay.[1.6]
*How does the CRISPR-Cas12a fluorescence assay work?*
When CRISPR-Cas12a binds to crRNA, they form an RNP complex (ribonucleoprotein complex). When the RNP
complex recognizes dsDNA that has complementary base-pairing to the guide crRNA, it acquires
non-specific ssDNA cleavage activity, collateral activity. If a probe composed of a fluorophore (FAM)
and a quencher connected by a 5-nucleotide sequence (TTATT) was cleaved by the activated Cas12a, it
displays increased fluorescence[1.7]. This CRISPR-Cas12a fluorescence assay
enhances specificity for
nucleic acid detection significantly[1.8].

![](https://static.igem.org/mediawiki/2021/4/4a/T--Kyoto--Engineering_004_Cas12a_2.png)

Build

We designed primers for the LAMP method of DMV[1.9].
In the design of crRNAs for CRISPR-Cas12a, the suitable sites for crRNAs were determined in the
sequences to be amplified by the RT-LAMP method[1.10].

Test

We performed CRISPR-Cas12a-mediated LAMP assay[1.11] against DMV using a
purchased B.
stearothermophilus
DNA polymerase, Bst3.0 (New England BIolabs). We made a series of dilution of DMV target DNA
(synthesized by IDT) from 1010 copies to 1 copy for the LAMP reaction to
verify the detection
limit. From the value of FAM, we confirmed that the detection limit is 1 copy.

![](https://static.igem.org/mediawiki/2021/9/97/T--Kyoto--Engineering_053.png)

![](https://static.igem.org/mediawiki/2021/4/47/T--Kyoto--Result_115_detection_saishin_.png)

Learn

Since we succeeded in CRISPR-Cas12a-mediated LAMP assay for DMV that is a DNA virus, we decided to
proceed to CRISPR-Cas12a-mediated RT-LAMP assay to detect other RNA viruses.

Cycle2
 We performed
CRISPR-Cas12a mediated RT-LAMP assay to
detect RNA viruses such as CSVd or TSWV using
purchased enzymes

Design

We reverse-transcribed RNA into cDNA, and then tried to detect RNA viruses such as CSVd and TSWV in
the same way as CRISPR-Cas12a mediated LAMP assay.

Build

We designed primers for RT-LAMP assay against CSVd and TSWV[1.12][1.13].
In the design of crRNAs for CRISPR-Cas12a, the suitable sites for crRNAs were determined in the
sequences to be amplified by the RT-LAMP method.

Test

In this experiment, we aimed to detect CSVd and TSWV using RNA samples obtained from actual infected
plants. We got these RNA samples from a collaborator. He kindly gave us two RNA samples of each.
Since BST polymerase 2.0 has reverse transcription activity, we used twice the amount of the enzyme
to perform the RT-LAMP reaction. Since we were told to dilute the samples 20 times, we prepared a
3-step gradient of samples: original concentration, diluted 20 times, and a no-template one. For
RT-LAMP reaction, samples in the following table were incubated at 63°C for 30 minutes.

![](https://static.igem.org/mediawiki/2021/2/20/T--Kyoto--Result_118_detecton.png)

After the assay, we checked the fluorescence of FAM with the naked eye under a blue light reader. We
also measured the value of FAM quantitatively by a qPCR machine, but as shown in the graph, sufficient
fluorescence could not be confirmed.

![](https://static.igem.org/mediawiki/2021/2/25/T--Kyoto--Result_119_detection.png)

We also performed Cas12a-mediated RT-LAMP assay on TSWV samples. For TSWV we prepared two types of
crRNA, and we performed the RT-LAMP reaction by incubating the samples in the following table at 63°C
for 30 min.

![](https://static.igem.org/mediawiki/2021/0/05/T--Kyoto--Result_120_detection.png)

After the assay, we checked the fluorescence of FAM with the naked eye under a blue light reader. We
also
measured the value of FAM quantitatively by a qPCR machine, but as shown in the graph, sufficient
fluorescence could not be confirmed this time ,either. The problems seem to be that the quality of the
RNA
samples was not confirmed and the optimal conditions for BST2.0 warm start were not sufficiently
examined,
so we would like to solve these in the future.

![](https://static.igem.org/mediawiki/2021/8/8e/T--Kyoto--Result_121_detection.png)

Learn

We found through Cycle1 and Cycle2 that a purchased BST DNA polymerase can be successfully used for
virus or viroid detection. However, the assay using purchased BST 3.0 / BST2.0 warmstart in the
recommended protocol would cost about $0.5 per reaction. Considering that the typical market price of
dahlia is about $7, it would be difficult for farmers to test all the crops and completely eliminate
the
spread of the viruses. Thus, in order to reduce the cost, we decided to produce enzymes by ourselves.

Cycle3
  We reduced the
cost of one reaction by producing homemade enzymes

Design

We considered producing the patent expired enzymes (BST DNA polymerase, Reverse Transcriptase) by
ourselves to lower the price per one reaction.

Build

We decided to prepare HIV-1 reverse transcriptase and B. stearothermophilus DNA polymerase. For HIV-1
RT, a peptide made from one ORF is partially digested in an infected cell and becomes two fragments,
p66
and p51. It is known that the HIV-1 RT functions when the p66 and p51 form a heterodimer. To purify
them
as recombinant proteins in E.coli, we separately expressed and purified p66 and p51, and dimerized
them
in vitro before use[1.14][1.15].

Test

We performed the purification of proteins in the following steps.

1. *Transformation*
   The genes of interest were cloned into pET-11a and were transformed into BL21 (DE3). A lot of
   colonies were observed in all the plates.
2. *Protein expression*
   Each LB culture was grown at 37 °C to the log-phase. When OD600 reaches 0.35-0.7, we added IPTG at
   the final concentration of 0.5 mM. After 3 hours of induction, cells were harvested and frozen
   in
   liquid-N2.
3. *Protein purification*
   After the cell disruption by sonication, each protein was purified using Ni-NTA beads. Samples
   were
   eluted by a four-step gradient of Imidazole. We observed the correct bands in the SDS-PAGE gel
   (shown below). The arrowheads indicate the targeted proteins.

![](https://static.igem.org/mediawiki/2021/f/f5/T--Kyoto--Result_102_detection.png)

4. *Dialysis*
   We put the purified proteins in dialysis bags and incubated them o/n at 4°C in 500 mL of storage
   buffer.
5. *Assay*
   We conducted the LAMP reaction against DMV with the purified homemade DNA polymerase. As shown in
   the figure below, we observed the ladder-like pattern, which is a typical pattern of LAMP
   reaction[1.16].
   We concluded that the LAMP reaction by homemade DNA polymerase is a success. Isothermal Buffer 1
   is a
   condition for increasing the DNA polymerase activity of BST3.0. Isothermal Buffer 2 is for
   increasing the reverse transcription activity of the enzyme.

![](https://static.igem.org/mediawiki/2021/3/36/T--Kyoto--Result_110_detection.png)

In addition to our homemade DNA polymerase, we examined our homemade reverse transcriptase activity in
vitro. We used total yeast DNA/RNA for this assay. We selected the intron-containing region of Rpl19A
as
a template for reverse transcription. If there is no reverse transcription, the genomic DNA including
an
intron is amplified by PCR, resulting in a band of 876 bp. On the other hand, if cDNA is correctly
synthesized by reverse transcription, a band of 370 bp without an intron should appear.
As shown below, the cDNA band was successfully observed in an RT-dependent manner. From this result,
we
concluded the activity of homemade RT[1.17].

![](https://static.igem.org/mediawiki/2021/4/45/T--Kyoto--Result_107_detection.png)

(From left to right: 1-4: newly prepared homemade RT with different enzyme concentrations, 5: no
enzyme,
6: BST3.0, 7: old homemade RT, 8: no template control)

Learn

We successfully produced the two enzymes necessary for RT-LAMP and confirmed their activities. We
reduced the cost of the reaction through Cycle3, which brought us one step forward to the practical
application of point-of-care detection at farms. However, there is one problem that we have to solve
for
practical application. That is, “how to measure the fluorescence generated by this assay”.

Cycle4
 Create hardware
that allows farmers to check detection results on site

Design

To address this problem, we decided to develop hardware that anyone can use easily and quickly. We
designed hardware that enables visual judgment.

Build

We designed the hardware named “DLAMI” (diseased leaves assessment, machine interface) for
fluorometry.

![](https://static.igem.org/mediawiki/2021/d/db/T--Kyoto--Engineering_005_Hardware_plan.png)

DLAMI hardware is a black box equipped PCR tube stand with a lid. There are two holes in
the sides of the box. The bigger hole is used for a 470 nm blue LED for the excitation of 5-FAM
fluorescence (maximum absorption wavelength at 495 nm, maximum emission at 520 nm)[1.18]. The smaller
hole
is
a camera hole by which we acquire the sample images. In the front of the camera hole, we inserted a
525
nm long-pass filter to cut off the irradiation light.

Test

DLAMI hardware was constructed as shown below.

![](https://static.igem.org/mediawiki/2021/e/ee/T--Kyoto--File_Proof_006.1.2_DLAMIofDLAMI.png)

DLAMI was tested with a PCR tube containing virus DNA template. As clearly shown in picture below, we
could take a picture of the tube with fluorescence by a camera of our smartphone. Thus, we concluded
that DLAMI can be used as a portable dark room for fluorescence detection by a smartphone.

![](https://static.igem.org/mediawiki/2021/8/88/T--Kyoto--File_DLAMI_fluor_1021.19.40.png)

Learn

We interviewed Mr. Kurokawa, who is a flower farmer in Kyoto, to confirm whether DLAMI hardware is
useful for farmers as we expected. We showed him the blueprint of DLAMI hardware and RT-LAMP assay.
We
received a number of feedback from him which were used to improve our system.

Cycle5
 Image diagnosis as a
pre-screening system

Design

So far we succeeded in creating a detection system that farmers can use on site. To improve this
system, we also developed an image diagnosis software to increase the probability of a positive
result
as a pre-screening. For tests involving nucleic acid amplification such as LAMP and PCR, it is
important to increase the prior probability of a positive result through pre-screening to minimize
false positives. For this purpose, we have created DLAEMON (Diseased Leaves Assessment by Efficient
Machine-learning On Neural network). This is an innovative diagnostic imaging software that allows
AI
to automatically diagnose diseases, by just taking an image of a leaf and clicking the diagnose
button. By using this software prior to CRISPR-Cas12a assisted RT-LAMP test, the probability of
pre-diagnosis is greatly increased, contributing to a significant reduction in false positives. We
also developed a related software NOBITA (Numericalization Of Brightness of Image for one Touch
Assessment) for the quantification of the fluorescence after Cas12a reaction.

Build

For DLAEMON, we used leaf images downloaded from the public dataset, plant village[1.19]. Since our
enzyme
detection method targets dahlia viruses, initially we would have preferred to use dahlia leaf data.
However, it was hard to collect a large amount of dahlia leaf data. We decided to use cherry blossom
leaves instead to build and validate the algorithm. This consisted of 1146 pieces of training data,
360 pieces of validation data (health:disease=5:5), and 400 pieces of test data
(health:disease=5:5).
Fine-tuning[1.20] was performed using the pre-trained Resnet18.

Test

The accuracy of DLAEMON was examined and proved to be 99.9% for the training data, 99.7% for the
validation data, and 99.8% for the test data.

![](https://static.igem.org/mediawiki/2021/e/e0/T--Kyoto--software_010and016_learning_curve_fine.png)

We also developed NOBITA, which automatically converts uploaded photos into black and white square
images and outputs the brightness of the clicked location. Pillow, an image processing library, is
used as the conversion tool.

![](https://static.igem.org/mediawiki/2021/b/b9/T--Kyoto--software_021_NOBITA_procedure.png)

![](https://static.igem.org/mediawiki/2021/e/e4/T--Kyoto--software_024_NOBITA_procedure5.png)

Learn

Regarding to DLAEMON, the accuracy is very good for all the training, validation and testing data. It
is
expected that further fine-tuning using the model developed in this study will make it easier to
develop
models for other plant leaves. This suggests that it is likely that we will be able to increase the
prior probability of experiments for detection and provide stronger support for experimental results
for
more plants in the future. However, we realize that the images in the data set used in this study were
carefully taken one by one, and the distribution is far from that of the actual images taken by
farmers.
Therefore, we need to collect more divergent image data in order to create a model that can be applied
to the field.
Regarding to NOBITA, the comparison with ImageJ shows that luminance can be extracted with very high
accuracy. However, there is room for improvement, too. It would be more user-friendly if it could
automatically identify the location of the samples and quantitate those areas automatically. It would
be
also important to determine a specific threshold of the fluorescence intensity by further
experiments.

Cycle6
 Feed forward loop
between image diagnosis and a portable
device for enzymatic test

Design

We built a portable detection device that integrates three components: an enzymatic method to detect
viral infection, hardware for fluorescence detection, and imaging software to increase the prior
probability. Using our system, newly acquired images of leaves are linked to the corresponding results
of (RT)-LAMP. These data sets should be in turn useful to improve DLAEMON software. As more image
information is accumulated through inspections, more practical machine learning that takes into
account
the blurring of photographic techniques in the field will become possible. Those feed forward loop
will
greatly contribute to improving the accuracy of diagnosis.
Eventually, DLAEMON software may reach an accuracy where a definitive diagnosis can be made without
examination by enzymatic reactions. To achieve this, the level of accuracy will need to be carefully
determined, taking into account factors such as the selling price of the flowers, the degree of
decline
in the value of the product due to the virus, and the speed of transmission of the virus.
As a prospect, we also plan to develop a drone equipped with this software. This drone will be
hovering
and taking thousands of photos of plant leaves and screening the infected ones in a short period of
time. The farmer only needs to visit the suspected areas of the field to perform the CRISPR-Cas12a
assisted LAMP / RT-LAMP assay with DLAMI hardware. We hope our system becomes the groundwork to
establish such
efficient agriculture.

References

1.1 (JAPANESE) 球根増殖コンソーシアム ダリアのウイルス・ウイロイド病 診断マニュアル
(2019)
http://www.pref.miyazaki.lg.jp/sogonogyoshikenjo/shigoto/nogyo/topix/documents/44689\_20190624121204-1.pdf
1.2 Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N., and Hase, T.
(2000) "Loop-mediated isothermal amplification of DNA", *Nucleic Acids Res. 28*, E63.
1.3 (2019a). From PCR to LAMP: The evolution of rapid-testing in food safety.
https://food-safety-news.3m.com/fsn/lamp-vs-pcr/
1.4 Zhang, Y., Odiwuor, N., Xiong, J., Sun, L., Nyaruaba, R.O., Wei, H., and Tanner, N.A. (2020)
"Rapid molecular detection of SARS-CoV-2 (COVID-19) virus RNA using colorimetric LAMP",
(*medRxiv*).
1.5 New England Biolabs Loop-Mediated Isothermal Amplification
https://international.neb.com/applications/dna-amplification-pcr-and-qpcr/isothermal-amplification/loop-mediated-isothermal-amplification-lamp
1.6 Broughton, J.P., Deng, X., Yu, G., Fasching, C.L., Servellita, V., Singh, J., Miao, X.,
Streithorst, J.A., Granados, A., Sotomayor-Gonzalez, A., et al. (2020) "CRISPR-Cas12-based detection
of SARS-CoV-2", *Nat. Biotechnol. 38*, 870–874.
1.7 Chen, J.S., Ma, E., Harrington, L.B., Da Costa, M., Tian, X., Palefsky, J.M., and Doudna, J.A.
(2018) "CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity",
*Science 360*, 436–439.
1.8 Nguyen, L.T., Smith, B.M., and Jain, P.K. (2020) "Enhancement of trans-cleavage activity of Cas12a
with engineered crRNA enables amplified nucleic acid detection", *Nat. Commun. 11*, 4906.
1.9 Iftikhar, R., Moyo, L., and Pappu, H.R. (2020) "A loop‐mediated isothermal amplification assay for
the detection ofDahlia mosaic caulimovirusin Dahlia (Dahlia variabilis)", *Ann. Appl. Biol. 176*,
203–209.
1.10 New England Biolabs FAQ: How do I design a guide RNA for use with EnGen Lba Cas12a?
https://www.neb.com/faqs/2018/05/03/how-do-i-design-a-guide-rna-for-use-with-engen-lba-cas12a
1.11 Ali, Z., Aman, R., Mahas, A., Rao, G.S., Tehseen, M., Marsic, T., Salunke, R., Subudhi, A.K.,
Hala, S.M., Hamdan, S.M., et al. (2020) "iSCAN: An RT-LAMP-coupled CRISPR-Cas12 module for rapid,
sensitive detection of SARS-CoV-2", *Virus Res. 288*, 198129.
1.12 Fukuta, S., Ohishi, K., Yoshida, K., Mizukami, Y., Ishida, A., and Kanbe, M. (2004) "Development
of immunocapture reverse transcription loop-mediated isothermal amplification for the detection of
tomato spotted wilt virus from chrysanthemum", *J. Virol. Methods 121*, 49–55.
1.13 Nagamine, K., Hase, T., and Notomi, T. (2002) "Accelerated reaction by loop-mediated isothermal
amplification using loop primers", *Mol. Cell. Probes 16*, 223–229.
1.14 Seckler, J.M., Howard, K.J., Barkley, M.D., and Wintrode, P.L. (2009) "Solution structural
dynamics of HIV-1 reverse transcriptase heterodimer", *Biochemistry 48*, 7646–7655.
1.15 https://biotechlink.org/1-2017/article6 (Link expired)
1.16 Lee, S.H., Baek, Y.H., Kim, Y.-H., Choi, Y.-K., Song, M.-S., and Ahn, J.-Y. (2016) "One-Pot
Reverse Transcriptional Loop-Mediated Isothermal Amplification (RT-LAMP) for Detecting MERS-CoV",
*Front. Microbiol. 7*, 2166.
1.17 Kellner, M.J., Ross, J.J., Schnabl, J., Dekens, M.P.S., Heinen, R., Grishkovskaya, I., Bauer, B.,
Stadlmann, J., Menéndez-Arias, L., Fritsche-Polanz, R., et al. (2020) "A rapid, highly sensitive and
open-access SARS-CoV-2 detection assay for laboratory and home testing", bioRxiv
1.18 OPTO SCIENCE Filter finder https://www.optoscience.com/maker/omega/finder/
1.19 Ali, A. PlantVillage Dataset. https://www.kaggle.com/abdallahalidev/plantvillage-dataset
1.20 Radenovic, F., Tolias, G., and Chum, O. (2019) "Fine-Tuning CNN Image Retrieval with No Human
Annotation", *IEEE Trans. Pattern Anal. Mach. Intell. 41*, 1655–1668.

- Click to visit -

* [Feeding RNAi for western flower thrips](#sctargetB-0)
* [Cycle1](#sctargetB-1)
* [Cycle2](#sctargetB-2)
* [Cycle3](#sctargetB-3)
* [Cycle4](#sctargetB-4)
* [References](#sctargetB-7)

Feeding RNAi for western flower thrips

*[Cycle1. Preparation of in vitro
transcript is expensive](#sctargetB-1)
[Cycle2. Preparation of dsRNA in
vivo](#sctargetB-2)
[Cycle3. Running a dsRNA uptake
assay in Frankliniella occidentalis](#sctargetB-3)
[Cycle4. Construction of a
practical insecticidal system for thrips](#sctargetB-4)*

Cycle1
  Preparation of in
vitro transcript is expensive.

Design

Currently, chemical pesticides are the most commonly used method to control
Frankliniella
occidentalis damage on flowers. However, many Frankliniella occidentalis have already
become
resistant to chemical pesticides.The damage caused by these tiny insects is increasing
year
by year.
We focused on feeding RNAi to kill these insects, based on previous studies showing that
oral ingestion of dsRNA can induce RNAi-induced gene knockdown in Frankliniella
occidentalis. To this end, we selected the essential gene vATPase-B in F. occidentalis,
as a
target of RNAi[2.1].
First, we planned to produce our dsRNA by in vitro transcription using the T7 RNA
polymerase.

Test

We transcribed several dsRNAs using a commercially available kit (Megascript kit,
Thermo).

Learn

However, in vitro transcript is still too expensive to be used as a pesticide. Also, as
the
yield of the system was not so high, we were not able to obtain the sufficient amount of
dsRNA for our experiments.

Cycle2
 Preparation of dsRNA
in vivo

Design

We decided to use in vivo dsRNA production system to obtain the sufficient amount of
dsRNA
for our experiments. To produce dsRNA in vivo, we used E. coli strain HT115 (DE3) and
plasmid vector L4440[2.2][2.3][2.4].
・HT115(DE3)
HT115 (DE3) is an RNase III-deficient E. coli strain that has been modified to express
T7
RNA polymerase from an IPTG-inducible promoter. This strain lacks RNase III which is a
dsRNA-specific nuclease. This mutation allows the strain to produce large amounts of
foreign
dsRNA.
・L4440

![](https://static.igem.org/mediawiki/2021/5/58/T--Kyoto--Engineering_RNA_201_sashikae.png)

L4440 is a plasmid vector having two inverted T7 promoters with a multi-cloning site in
between. A part of the target gene is inserted into the multi-cloning site. The target
sequence is transcribed from both sides, so dsRNA can be obtained when both transcripts
anneal.

Build

We created a series of plasmids which contain a target gene sequence between two T7
promoters.
To this end, L4440 was cut with restriction enzymes and the target gene fragment was
inserted by
DNA ligation.

Test

We constructed 3 plasmids for dsRNA production. Using these plasmids, we expressed 3 dsRNAs
in
HT115(DE3) in parallel.
We tested two different protocols for the purification[2.5]. To estimate
the amounts of dsRNA
recovered in each round, purified nucleic acids were separated by 8% PAGE.

![](https://static.igem.org/mediawiki/2021/9/98/T--Kyoto--Engineering_105_RNAgel1.jpg)

(1. vATPase-B 1, 2. vATPase-B 2, 3. EPH1, 4. L4440, 5. vATPase-B 1, 6. vATPase-B 2, 7. EPH1, 8.
L4440)

![](https://static.igem.org/mediawiki/2021/3/37/T--Kyoto--Engineering_105_1_rnagel2.jpg)

(1. L4440 (add production) (1ug), 2. vATPase-B (add production) (7ug), 3.
vATPase-B (add
production) (1ug ), 4. vATPase-B-1 (1ug), 5. vATPase-B-2 (1ug), in vitro RNA transcription (6.
vATPase-B,7. Trxz,8 EPH1 ) 1ug each)

In the first round of production, we observed a major band of vATPase-B around 700 bp, which
corresponds to the target product length. The band visible in EPH1 is slightly shorter than
that
of vATPase-B, corresponding to the shorter length of the product. In summary, we obtained a
certain amount of the target dsRNA in this preparation.

![](https://static.igem.org/mediawiki/2021/0/08/T--Kyoto--Engineering_106_Table_RNA_purification_v4.png)

In the second round of trials, the yield of each dsRNAs was much higher than the previous
samples. This could be because the purification protocol was simplified at this time. We
noticed that a certain amount of samples could be lost during preparation.

Cycle3
  Running a dsRNA
uptake assay in Frankliniella
occidentalis

Design

Using our dsRNA sample which targets vATPase-B gene of thrips, we
tested the insecticidal effect of feeding RNAi.
Previous research reported that thrips can be killed by feeding dsRNA of the
same gene as we used. However, in the paper, the authors used purified in vitro
transcripts as dsRNAs. As we prepare our dsRNA in vivo, our RNA contains a lot of host
RNA species including tRNAs and rRNAs. We examined the effect of these
contaminated materials on the feeding RNAi assay.
"RNAi" (RNA interference) is a process in which externally introduced dsRNA suppresses the
expression of genes that have complementary sequences to the dsRNA[2.6].

1. dsRNA is taken in from the outside.
2. An enzyme called Dicer cleaves dsRNA to form a short RNA strand called siRNA.
3. The siRNA forms a complex called RISC (RNA-induced silencing complex) with proteins
   such as Argonaute protein.
4. Target genes are silenced.

"vATPase-B"
vATPase-B is a gene that encodes a protein that is part of a subunit of vATPase, a type
of ATP-dependent proton pump that regulates the pH of various cell organelles. Since
this gene is essential for thrips, knockdown of vATPase-B results in the death of the
thrips.

Build

Pipette 200 µl of 200 ng/ul dsRNA solution targeting vATPase-B (Milli-Q was used as a negative
control) into a PCR tube and soak the petiole of the green bean primary leaf. 2 hours later, cut three
1 cm × 2 cm leaf pieces from the soaked green bean leaf and place them in three petri dishes with
about 10 thrips. We checked the mortality at 24, 48, 72, and 96 hours[2.1][2.7][2.8]

Test

![](https://static.igem.org/mediawiki/2021/5/52/T--Kyoto--Proof_008_RNAi2.png)

In particular, there was a large difference in the average mortality rate between
dsRNA-treated and negative control thrips on the third day, which was 8.3% and 31.3%,
respectively. This result confirms the insecticidal effect of indirectly feeding dsRNA
to western flower thrips. We also found that the use of the crude dsRNA samples were as
effective as the reported study with the T7 transcripts.

Learn

Unfortunately, the method examined here did not result in a dramatically high level of
the insecticidal effect. One possible explanation is that the concentration of the dsRNA
solution used in the experiment was too low. We need a more efficient dsRNA production
system.

Cycle4
 Construction of a
practical insecticidal system for
thrips

Design

When considering the practical use of a highly concentrated dsRNA solution as an
insecticide, we can take advantage of the scale merit of mass production. In this case,
our efficient biomolecule production platform "BLOOM" can be used for this purpose [(see here for details)](https://2021.igem.org/Team%3AKyoto/Implementation)
Alternatively,
it would be also a promising option to kill thrips if we create
transgenic plants that continuously express dsRNA. Our results described above will
provide us a smart system to select the most effective dsRNA species for this
application.

References

2.1 Andongma, A.A., Greig, C., Dyson, P.J., Flynn, N., and Whitten, M.M.A. (2020) "Optimization of
dietary RNA interference delivery to western flower thrips Frankliniella occidentalis and onion thrips
Thrips tabaci", *Arch. Insect Biochem. Physiol. 103*, e21645.
2.2 Tenllado, F., Martínez-García, B., Vargas, M., and Díaz-Ruíz, J.R. (2003) "Crude extracts of
bacterially expressed dsRNA can be used to protect plants against virus infections", *BMC
Biotechnol. 3*,
2.3 Timmons, L., and Fire, A. (1998) "Specific interference by ingested dsRNA", *Nature 395*,
854.
2.4 https://2019.igem.org/Team:SZU-China/Experiment
2.5 Papić, L., Rivas, J., Toledo, S., and Romero, J. (2018) "Double-stranded RNA production and the
kinetics of recombinant Escherichia coli HT115 in fed-batch culture", *Biotechnol Rep (Amst) 20*,
e00292.
2.6 Limera, C., Sabbadini, S., Sweet, J.B., and Mezzetti, B. (2017) "New Biotechnological Tools for
the Genetic Improvement of Major Woody Fruit Species", *Front. Plant Sci. 8*, 1418.
2.7 Han, S.H., Kim, J.H., Kim, K., and Lee, S.H. (2019) "Selection of lethal genes for ingestion RNA
interference against western flower thrips, Frankliniella occidentalis, via leaf disc-mediated dsRNA
delivery", *Pestic. Biochem. Physiol. 161*, 47–53.
2.8 Singh, S., Gupta, M., Pandher, S., Kaur, G., Goel, N., Rathore, P., and Palli, S.R. (2019) "RNA
sequencing, selection of reference genes and demonstration of feeding RNAi in Thrips tabaci (Lind.)
(Thysanoptera: Thripidae)", *BMC Mol. Biol. 20*, 6.

- Click to visit -

* [Other applications for cut flower maintenance](#sctargetC-0)
* [Cycle1](#sctargetC-1)
* [Cycle2](#sctargetC-2)
* [Cycle3](#sctargetC-3)
* [Cycle4](#sctargetC-4)
* [Cycle5](#sctargetC-5)
* [Cycle6](#sctargetC-6)
* [References](#sctargetC-7)

Other applications for cut flower maintenance

We have been investigating various problems in the production, distribution and consumption of
flowers.
Our research has focused on the detection of plant viruses in the production stage. At the same time,
we
also focused on the problem of cut flowers in our daily lives.
*[Cycle1. Commercially available peptide is
expensive](#sctargetC-1)
[Cycle2. Preparation of peptide in vivo](#sctargetC-2)
[Cycle3. Improve how to preparate of peptide in
vivo](#sctargetC-3)
[Cycle4. Run an assay to see how long the flowers
last](#sctargetC-4)
[Cycle5. Pre-research for measure ability of
peptide to kill bacteria](#sctargetC-5)
[Cycle6. Disk diffusion test to measure the
ability
of the peptide to kill bacteria](#sctargetC-6)*

Cycle1.
 Commercially
available peptide is expensive

Design

We focused on "NOP-1", which is derived from the NLS of ethylene regulatory factor EIN2, which can
inhibit the senescence signal of flowering plants by acting on ethylene receptors[3.1][3.2].
Currently, the silver thiosulfate complex (STS) is commonly used to extend the life of cut flowers.
However, STS contains silver ion, which is a heavy metal ion, and there are concerns about its
negative
impact on the environment, so there is a need to develop a new alternative method.
One of the causes of cutflowers wilter is the growth of bacteria in the water in the vase, which
blocks the conduits of the flowers and prevents the flowers from absorbing water[3.3]. This is not only a
big
problem at the distribution and sales stage, but also at home.
To solve this problem, we focused on the application of antimicrobial peptides, which have a low
environmental impact, instead of antibiotics or chemicals. Antimicrobial peptides act on bacterial
cell
membranes and kill the bacteria by puncturing the membrane. These peptides have been found to be
effective against many bacteria[3.4].
For this experiment, we selected the following three antimicrobial peptides.
*Defensin-1*
It is an antimicrobial peptide derived from honey bees and is known to be widely effective against
Gram-positive bacteria[3.5][3.6].
potent antibacterial protein in royal jelly. Purification and determination of the primary structure
of
royalisin.
*Cecropin A*
It is a peptide derived from a moth called Cecropia, which is known to be widely effective against
both
Gram-positive and Gram-negative bacteria[3.7].
*LL37*
It is a human-derived peptide that is known to be widely effective against both Gram-positive and
Gram-negative bacteria[3.8][3.9].
Susceptibilities of periodontopathogenic and cariogenic bacteria to antibacterial peptides,
β-defensins
and LL37, produced by human epithelial cells

The first step was to purchase a chemically synthesized peptide to test the effect of this
antimicrobial
peptide on flower longevity.

Learn

However, chemically synthesized peptides were deemed too expensive to be used in experiments.

Cycle2.
  Preparation of
peptide in vivo

Design

Therefore, we decided to express NOP-1 and three antimicrobial peptides in E. coli and purify them.
The
paper was used as a reference to produce the peptide[3.10].
The following method was used[3.11]. The peptide is expressed as a fusion
protein: peptide-Mxe GyrA
intein-linker-ELK16. The fusion protein aggregates and precipitates due to the action of ELK16. When
DTT
is added to the precipitated fusion protein, self-cleavage occurs at the N-terminus of the intein, and
the peptide is cut out into the water layer.

![](https://static.igem.org/mediawiki/2021/e/e3/T--Kyoto--Engineering_200_ELK16.png)

*ELK16*
It is an ionic self-assembling peptide that promotes the formation of active inclusion bodies and
active
protein aggregates in *E. coli*, and because it self-aggregates, the fusion protein can be
recovered as
a
precipitate.
*Mxe GyrA intein*
A sequence that is autocatalytically cleaved during protein splicing. By fusing it downstream of the
peptide, the peptide can be expressed as a fusion protein, and by adding DTT as a reducing agent, the
activity of the intein can be evoked for self-cleavage.

Build

We made the following plan. First, we inserted the gblock encoding the fusion protein into pET11a to
make a plasmid for peptide production. The plasmid was then transformed into *E. coli BL21*, and
peptide
production was carried out as described in the design.

![](https://static.igem.org/mediawiki/2021/c/cc/T--kyoto--plasmid_ELK16.png)

Test

After making the plasmid, we produced peptides and cut out the peptides from fusion proteins. The
recovered peptides were electrophoresed on Tricine SDS PAGE to determine whether the peptides were
produced.

![](https://static.igem.org/mediawiki/2021/a/a4/T--Kyoto--Engineering_peptide_sashikae1.png)

1~12 1a~4a,1b~4b,1s~4s (a-sonication 10sx2 ,b-sonication 10s x2 + bioruptor 2sx100, c-sonication
10s x2
+
2sx100)

Learn

The band that is considered to be Def1(5713 Da) appears, while it could not be confirmed as a major
band in the rest of the peptides. Also, the molecular weight of the fusion protein of intein and ELK16
is about 25 kDa, and this band is also visible. It seems that the impurities have been reduced by
additional sonication, but still many impurities remained.

Cycle3
 Improve how to
preparate of peptide in vivo

Design

There are three problems that we found during the first round of peptide purification. First, when the
fusion protein is recovered as a precipitate using ELK16, the purified peptide contains many
impurities
even after washing. Second, the precipitates contain highly viscous material such as genomic DNA,
which
makes it difficult to repeat peptide production because of the time required to wash the
precipitates is long. Third, peptides other than Def1 cannot be identified as major bands, and the
amount
recovered is quite low. To solve this problem, we decided to change the ELK16 part to a His-tag, and
purify the fusion protein of the target peptide and intein by His-tag purification, and then recover
the
peptide by activating the intein by addition of DTT.
The effect of n-end-rule was also considered as the cause of the problem of low recovery[3.12]. I thought
that
this might have been the reason for the low recovery of peptides other than Def1, so we designed to
improve the N-terminal of the peptides. Since NOP1 is originally part of a protein, we will create a
plasmid to produce a peptide with valine added to the N-terminus of NOP1.

Build

Using an additional primer, a DNA fragment of His tag-intein-peptide was amplified by PCR from the
gblock of ELK16-intein-peptide. Then, we inserted the created fragment into pET11a to make plasmid for
peptide production and purification by His tag. We transformed these plasmids into BL21 and poducted
peptide using the method described in Design.

Test

After plasmids construction was completed, we produced fusion protein in BL21 and pulificated by His
tag.The peptides were electrophoresed by Tricine SDS PAGE.

![](https://static.igem.org/mediawiki/2021/c/c0/T--Kyoto--Engineering_peptide_sashikae2.png)

(1 CecA 2 Def1 3 LL37 4 NOP1 5 NOP1v 6 pet11a 7 CecA 8 Def1 9 LL37 10 NOP1 11
NOP1v)

the bands of the peptides could be seen in Def1 with and without DTT. In all lanes except pet11a, a
band
(of 24 kDa) that seems to be His-intein were visible. Therefore, the expression of the fusion protein
and
the purification of the fusion protein using His tag seems to have been successful. There are three
possible reasons why the other peptides were not identified as bands. First, NOP1 is a particularly
short peptide and may have run away during electrophoresis. The second possibility is that the
peptides were not cleaved by the self-cleavage of the intein, and the third possibility is that the
intein was cleaved at some point before purification and moved to another fraction during imidazole
elution. Def1 was also cut out before the addition of DTT, so this may have occurred the same with
other
peptides.

Cycle4.
 Run an assay to see
how long the flowers last

Design

An existing method, STS, competitively inhibits ethylene binding to Cu+ in the ethylene receptor
(ETR1)
of flowering plants with Ag+, which also has a high affinity for ethylene. Ag+ is sterically larger
than
Cu+ and occupies the majority of the binding site, allowing it to bind to ethylene and inhibit
ethylene
binding to Cu[3.13].
In contrast, NOP-1 is a peptide known to prolong the life of flowering plants by a mechanism different
from that of STS, and can inhibit ethylene-dependent senescence. The signaling pathway triggered by
the
binding of ethylene to ETR1 inactivates CTR1 kinase and inhibits the phosphorylation of the ethylene
regulatory factor EIN2, thereby activating the expression of ethylene response genes. Therefore,
NOP-1,
a peptide derived from the nuclear localization signal of EIN2, can regulate senescence signaling by
binding to the GAF domain of ETR1 and arresting intra- and intermolecular downstream signaling of the
receptor. In fact, it was confirmed that flower senescence of flowering plants treated with NOP-1 was
suppressed[3.14][1.15].

![](https://static.igem.org/mediawiki/2021/0/0c/T--Kyoto--Engineering_205_Ethylene_pathway.pptx.png)

![](https://static.igem.org/mediawiki/2021/b/b0/T--Kyoto--Engineering_205_Ethylene_pathway2.pptx.png)

Build

We tested the effects of (1) NOP-1 and NOP-1v synthesized in vivo, (2) Brassinolide and Brassinazole
given from Mr.Nakano, (3) NOP-1 synthesized chemically. To avoid the possibility of prior treatment
with
STS agents, we used ethylene-sensitive flowering plant, Dianthus, which was purchased in potted form.
With reference to previous studies, 1 mM AgNO3 was used as a positive control and the same buffer in
which each peptide was dissolved was used as a negative control.

Test

No significant flower life extension effect was observed for both NOP-1 synthesized in vivo and NOP-1
synthesized chemically.

Learn

From the literature[3.16], it is known that NOP-1 peptide acts on ethylene
receptors in the endoplasmic
reticulum membrane to prolong the life of cut flowers. In this study, due to seasonal and cost issues,
we used Dianthus instead of roses and carnations, which were used in previous studies. Therefore, the
results of this experiment indicate that in Dianthus, NOP-1 peptide may not reach the ethylene
receptors
of the endoplasmic reticulum membrane in sufficient amounts.

Cycle5.
 Pre-research for
measuring the ability of peptide to
kill
bacteria

Design

One of the causes of cut flowers wilter is the growth of bacteria in the water in the vase, which
blocks the conduits of the flowers and prevents the flowers from absorbing water[3.17]. This is not only a
big
problem at the distribution and sales stage, but also at home.
To address this issue, we searched the literature to learn more about the fungal flora in vases.
However, we could not find satisfactory previous studies on the fungal flora in vases. Therefore, we
decided to investigate what kind of fungi exist in a vase first.

Build

We grew rose, carnation, and delphinium for one week. We had taken bacteria from the vase surface,
stem,
and vase water by platinum loop and smeared on the standard agar medium (Agar 15g,
Glucose 1g, peptone 6g, and Yeast extarct2.5g per 1L water ). In addition, we had collected water in
each place and spread it or its diluted solution on the standard agar medium uniformly by conlarge
stick. This method is appropriate to observe colonies. To compare these plates, we thought that we
could
understand the changes in bacterial flora that depend on time, place, and spices of flowers in the
vase.

Test

The bacterial flora of rose and delphinium had changed over time dramatically. Besides, these flowers
wilted. We could not observe the changes in bacterial flora of carnation as clearly as rose and
delphinium. However, the vase water of carnation cultivated the most amount of bacteria.

Learn

When we observed the changes in bacterial flora, pH also changed. So, the bacteria growing each day
could
not live without the distinctive environment of the day. The dominant species would change as the day
passed[3.18]. One of the major causes of these changes might be that the
dominant species growing rapidly
would decompose the nutrients that plants excrete as a result of the stress reaction or seep out
of
the phloem[3.19][3.20]. This would eliminate waste products, which might result in the continuous environmental
change
in the vase. From these considerations, we thought that if we could always inhibit the bacteria that
might grow in the early stage, we would stop the environmental change. As a result, this would keep
water clean and cut flowers beautiful. In addition, we had researched the bacterial flora in the
stem,
so that we could identify the bacteria that might prevent the vessel from absorbing water. It will be
more
effective to kill the bacteria if we can understand its traits or weak points. If we could do these
experiments, we would be able to change water less often and clean waste water.

Cycle6.
 Disk diffusion test
to measure the ability of the
peptide to kill bacteria

Design

To measure the activity of peptide purified by His-tag in cycle3, we decided to do the disk diffusion.

Build

From pre-research, We found that we were able to measure the activity of Defensin and CecropinA by
using
Bacillus subtilis and E.coli[3.21][3.22][3.23].

Test

We did the disk diffusion method to check if the antimicrobial peptides we made have the ability to
kill
the bacteria[3.24]. In this experiment, we used Escherichia coli and
Bacillus subtilis. However, no antimicrobial activity was observed by our peptides.

Learn

The possible reason for the lack of antimicrobial activity might be that the concentration
of
antimicrobial peptides was not sufficient or the number of bacteria was so large that their activity
could not work sufficiently.

References

3.1 Hoppen, C., Müller, L., Albrecht, A.C., and Groth, G. (2019) "The NOP-1 peptide derived from the
central regulator of ethylene signaling EIN2 delays floral senescence in cut flowers", *Sci. Rep.
9*, 1287.
3.2 Kessenbrock, M., Klein, S.M., Müller, L., Hunsche, M., Noga, G., and Groth, G. (2017) "Novel
Protein-Protein Inhibitor Based Approach to Control Plant Ethylene Responses: Synthetic Peptides for
Ripening Control", *Front. Plant Sci. 8*, 1528.
3.3 Kessenbrock, M., Klein, S.M., Müller, L., Hunsche, M., Noga, G., and Groth, G. (2017) "Novel
Protein-Protein Inhibitor Based Approach to Control Plant Ethylene Responses: Synthetic Peptides for
Ripening Control", *Front. Plant Sci. 8*, 1528.
3.4 Zhang, L.-J., and Gallo, R.L. (2016) "Antimicrobial peptides", *Curr. Biol. 26*, R14–R19.
3.5 Fujiwara, S., Imai, J., Fujiwara, M., Yaeshima, T., Kawashima, T., and Kobayashi, K. (1990) "A
potent antibacterial protein in royal jelly. Purification and determination of the primary structure
of royalisin", *J. Biol. Chem. 265*, 11333–11337.
3.6 Defensin1 http://parts.igem.org/Part:BBa\_K1104301
3.7 Steiner, H., Andreu, D., and Merrifield, R.B. (1988) "Binding and action of cecropin and cecropin
analogues: antibacterial peptides from insects", *Biochim. Biophys. Acta 939*, 260–266.
3.8 Ouhara, K., Komatsuzawa, H., Yamada, S., Shiba, H., Fujiwara, T., Ohara, M., Sayama, K.,
Hashimoto, K., Kurihara, H., and Sugai, M. (2005). "Susceptibilities of periodontopathogenic and
cariogenic bacteria to antibacterial peptides, {beta}-defensins and LL37, produced by human epithelial
cells", *J. Antimicrob. Chemother. 55*, 888–896.
3.9 LL-37 antimicrobial peptide http://parts.igem.org/Part:BBa\_K1162006
3.10 Wang, M., Zheng, K., Lin, J., Huang, M., Ma, Y., Li, S., Luo, X., and Wang, J. (2018) "Rapid and
efficient production of cecropin A antibacterial peptide in Escherichia coli by fusion with a
self-aggregating protein", *BMC Biotechnol. 18*, 62.
3.11 Lin, Z., Zhao, Q., Zhou, B., Xing, L., and Xu, W. (2015) "Cleavable Self-Aggregating Tags (cSAT)
for Protein Expression and Purification. In Insoluble Proteins: Methods and Protocols, E.
García-Fruitós, ed", (*New York, NY: Springer New York*), pp. 65–78.
3.12 Tobias, J.W., Shrader, T.E., Rocap, G., and Varshavsky, A. (1991) "The N-end rule in bacteria",
*Science 254*, 1374–1377.
3.13 McDaniel, B.K., and Binder, B.M. (2012) "ethylene receptor 1 (etr1) Is Sufficient and Has the
Predominant Role in Mediating Inhibition of Ethylene Responses by Silver in Arabidopsis thaliana",
*J. Biol. Chem. 287*, 26094–26103.
3.14 Hoppen, C., Müller, L., Albrecht, A.C., and Groth, G. (2019) "The NOP-1 peptide derived from the
central regulator of ethylene signaling EIN2 delays floral senescence in cut flowers", *Sci. Rep.
9*, 1287.
3.15 Binder, B.M. (2020) "Ethylene signaling in plants", *J. Biol. Chem. 295*, 7710–7725.
3.16 Hoppen, C., Müller, L., Albrecht, A.C., and Groth, G. (2019) "The NOP-1 peptide derived from the
central regulator of ethylene signaling EIN2 delays floral senescence in cut flowers", *Sci. Rep.
9*, 1287.
3.17 Florack, D.E.A., Stiekema, W.J., and Bosch, D. (1996) "Toxicity of peptides to bacteria present
in the vase water of cut roses", *Postharvest Biol. Technol. 8*, 285–291.
3.18 Simek, K., Pernthaler, J., Weinbauer, M.G., Hornák, K., Dolan, J.R., Nedoma, J., Masín, M., and
Amann, R. (2001) "Changes in bacterial community composition and dynamics and viral mortality rates
associated with enhanced flagellate grazing in a mesoeutrophic reservoir", *Appl. Environ.
Microbiol. 67*, 2723–2733.
3.19 Ratzke, C., and Gore, J. (2018). Modifying and reacting to the environmental pH can drive bacterial interactions. *PLoS Biol.* 16, e2004248.
3.20 Albert, L.S., and Brown, D.G. (2015). Variation in bacterial ATP concentration during rapid changes in extracellular pH and implications for the activity of attached bacteria. *Colloids Surf. B Biointerfaces* 132, 111–116.
3.21 Shen, L., Liu, D., Li, M., Jin, F., Din, M., Parnell, L.D., and Lai, C.-Q. (2012) "Mechanism of
action of recombinant acc-royalisin from royal jelly of Asian honeybee against gram-positive
bacteria", *PLoS One 7*, e47194.
3.22 Silvestro, L., Weiser, J.N., and Axelsen, P.H. (2000) "Antibacterial and antimembrane activities
of cecropin A in Escherichia coli. Antimicrob", *Agents Chemother. 44*, 602–607.
3.23 Oren, Z., Lerman, J.C., Gudmundsson, G.H., Agerberth, B., and Shai, Y. (1999) "Structure and
organization of the human antimicrobial peptide LL-37 in phospholipid membranes: relevance to the
molecular basis for its non-cell-selective activity", *Biochem. J 341 ( Pt 3)*, 501–513.
3.24 Tenover, F.C. (2009) "Antibiotic Susceptibility Testing. In Encyclopedia of Microbiology (Third
Edition), M. Schaechter, ed", (Oxford: Academic Press), pp. 67–77.

- Click to visit -

* [Biomolecular Production Platform](#sctargetD-0)
* [Cycle1](#sctargetD-1)
* [Cycle2](#sctargetD-2)
* [Cycle3](#sctargetD-3)
* [Cycle4](#sctargetD-4)
* [Cycle5](#sctargetD-5)
* [Cycle6](#sctargetD-6)
* [Cycle7](#sctargetD-7)
* [References](#sctargetD-8)

Biomolecular Production Platform

*[Cycle1. Model of APP using usual approaches](#sctargetD-1)
[Cycle2. Model of APP by adopting a stochastic
approach](#sctargetD-2)
[Cycle3. Reproduction of 2 plasmid system using
BLOOM](#sctargetD-3)
[Cycle4. ssrA tag mutagenesis](#sctargetD-4)
[Cycle5. To derive production efficiency in a
production system using BLOOM](#sctargetD-5)
[Cycle6. Reproduction of 3 plasmid system using
BLOOM](#sctargetD-6)
[Cycle7. Plasmid construction](#sctargetD-7)*

Cycle1.
 Model of APP using
usual approaches

Design

The efficient production of biomolecules is an important issue for the entire synthetic biology
community. Therefore, we have developed an intelligent biomolecule production system based on
asymmetric
plasmid partitioning.
Multicellular organisms produce functions that cannot be achieved by unicellular organisms through the
differentiation and division of roles among different types of cells. We aimed to give *E. coli*
the
ability to differentiate and divide its role by using asymmetric plasmid partitioning, and then use
this
ability to innovate biomolecular production systems.
*Asymmetric plasmid partitioning(APP)*
Plasmids are normally distributed equally among daughter cells. However, in asymmetric plasmid
partitioning, a particular plasmid is distributed to only one daughter cell.
This results in two cell types at each division, one with the plasmid and one without.
The specific mechanism is as follows[4.1].

![](https://static.igem.org/mediawiki/2021/2/2c/T--Kyoto--Modeling_APP.png)

The two actors are a protein called ParB and a DNA sequence on the plasmid is called ParS. ParS
attaches
to
ParB, and ParB has the property of self-aggregating. Therefore, the plasmid carrying ParS aggregates
under ParB expression, forming a nucleus-like aggregate that can only be distributed to one daughter
cell.

Build

First, we tried to build the first model to understand APP, and tried to simulate APP.
To get started, we classified *E.coli* as either “stem cells” or “differentiated cells”, and
kinetically calculated averaged plasmid copy numbers of them to reproduce dynamics of the overall
system.

Test

The first model demonstrated only a simple result. It cannot be said the model succeeded in
reproducing
APP.

Learn

Thus, we found that we cannot accurately reproduce APP by using only kinetics, and thought building a
model by using other approaches.

Cycle2.
  Model of APP by
adopting a stochastic approach

Build

We determined to use a stochastic approach in order to reproduce APP, selected “Gillespie
algorithm”[4.2].
We
decided not to simulate dynamics of the overall system or rather, to simulate dynamics of one cell,
and repeated
sufficient times to get the transition of dynamics of the system.

Test

We succeeded in reproducing the change of plasmid copy numbers shown in the graph below. We reproduced
the
result close to the thesis by adjusting the parameters assumed.

![](https://static.igem.org/mediawiki/2021/6/65/T--Kyoto--modeling_Modeling_02_01_2p_copy.png)

Learn

This result suggested that the Gillespie algorithm is efficient. Therefore, we thought that we can
simulate the transition of the concentration of contents based on this model.

Cycle3.
  Reproduction of 2
plasmid system using BLOOM

Design

We also thought that it would be more convenient if multiple genes could be expressed in a stepwise
manner in differentiated cells, as in the developmental process of multicellular organisms. Therefore,
we
decided to combine asymmetric plasmid partitioning with two repressor systems to express multiple
genes
in differentiated cells with a time delay.
The specific system is as follows.

![](https://static.igem.org/mediawiki/2021/d/de/T--Kyoto--Modeling_APP2.png)

1. All cells have both of the two plasmids and are in a stem cell type state. The two genes, GFP
   and
   RFP, are both repressed by repressors. However, due to the difference in promoters, there is a
   difference in concentration between the two repressors.
2. When arabinose is added, pBAD is turned on and parB expression is induced.
3. The plasmid carrying parS aggregates, and at the next division, asymmetric plasmid partitioning
   occurs and the plasmid carrying parS drops out from one of the daughter cells, resulting in a
   differentiated cell.
4. In cells that have lost the parS plasmid, no additional repressor production occurs, and the
   repressor concentration decreases.
5. When the concentration of one of the repressors falls below the threshold for repression, the
   first gene begins to expresing. In this prototype, we have used GFP as the first gene.
6. Furthermore, when the concentration of the other repressor falls below the threshold for
   expression repression, the second gene begins expresing. In this prototype, the second gene
   is
   RFP.
7. Thus, stepwise gene expression occurs in differentiated cells, and the daughter cells of the one
   that did not shed the plasmid return to (2), so that this cycle is sustained semi-permanently.

Build

We built a model of the 2 plasmid system. In addition, we investigated what is the most effective
parameter for customizing the time difference by observing the dynamics if we changed a parameter.

Test

The result is as follows.

![](https://static.igem.org/mediawiki/2021/3/32/T--Kyoto--modeling_Modeling_02_03_04_2p_ara%3D0.200_prod.png)

Please refer to the [Modeling page](https://2021.igem.org/Team%3AKyoto/Model) for variation
by degradation rate and promoter strength.

Learn

From the graphs, we found that BLOOM can generate the time difference in expressions of two kinds of
genes, and that the degradation rate of repressors is the most efficient for the time difference.
In addition, because the difference of the promoter activities of repressors makes the difference, we
told our wet team the promoter set that generates the time difference maximumly.

Cycle4.
 ssrA tag
mutagenesis

Design

To adapt our BLOOM system to various needs of biomolecule production, we focused on controlling the
time interval between the expression of two genes. Our mathematical modeling shows that the most
important parameter regulating the temporal difference of our BLOOM system is the half-life of
the repressors.
To control the protein half-life, we used ssrA degradation tag. In Escherichia Coli, ssrA-tagged
proteins are recognized and efficiently degraded by proteases such as ClpXP or ClpAP. A previous
research has identified 12 mutant ssrA tags that promote the degradation of GFP at different
rates[4.3]. Therefore, suspecting that there should be more mutants of
various degradation efficiencies, we aimed to identify them by mutagenizing WT tag.

Build

The wildtype ssrA tag amino acid sequence is AANDENYALAA. It was reported that the three amino acids
at the C terminus of the tag (LAA in wildtype) play a important role on the degradation rate of tagged
protein. Therefore, we chose these three amino acids as the targets for mutagenesis.
For mutagenesis, we used a primer containing three random bases at either one of the target three
C-terminal amino acids (XAA, LXA, LAX), or nine random bases at all of the target three amino acids
(XXX). We also included a primer containing wildtype sequence (LAA), and two reported mutants (AAV &
ASV) for controls.

![](https://static.igem.org/mediawiki/2021/e/e9/T--Kyoto--ssrA_005.png)

We fused the ssrA tag sequence to GFP while introducing mutations in the tag by
random-base primers, and cloned the mutant library of ssrA-tagged GFP into a plasmid vector, so that
mutant tags of different activity can be identified by comparing GFP intensity of *E.coli*
transformants.

![](https://static.igem.org/mediawiki/2021/7/78/T--Kyoto--TKG.png)

Our mutagenesis strategy is as follows:

1. to J23119 promoter-RBS-GFP insert derived from BBa\_K584001, add ssrA tag sequence lacking the
   three C-terminal amino acids by PCR (first PCR)
2. perform the second PCR on the first PCR product to add the rest of the tag sequence while
   mutagenizing them with different types of random-base primers listed above.
3. assemble the second PCR product with a linearized pSB4K5-BBa\_J04450 backbone by InFront Assembly
   (a type of homology arm-mediated seamless cloning like In-Fusion) and transform it into
   *E.coli*.

We chose a low-copy plasmid pSB4K5 as the backbone to be able to compare the
fluorescence intensity by trying to avoid the saturation or variation of GFP intensity of colonies.

![](https://static.igem.org/mediawiki/2021/3/36/T--Kyoto--Result_003_ssrA.png)

Test

As a result of transformation, colonies of various fluorescence intensity were obtained on the LB
plates. 72 colonies in total were picked from plates, and cultured overnight in LB media. The images
of *E.coli* cultures were taken under the blue light.
(plate images: under construction)

![](https://static.igem.org/mediawiki/2021/2/22/T--Kyoto--ssrA_007.png)

![](https://static.igem.org/mediawiki/2021/2/26/T--Kyoto--ssrA_011.png)

Bacterial cultures of various GFP intensity were observed.
A10: LB media only, B10: *E.coli* culture not expressing GFP, B7: *E.coli* culture
expressing non
ssrA-tagged GFP, C7: *E.coli* culture expressing WT ssrA-tagged GFP
Plasmid was extracted from
each
bacterial culture by miniprep, and then
Sanger-sequenced to identify ssrA tag sequence. Furthermore, to quantify protein degradation
efficiency of each mutant, GFP Fluorescence of each bacterial overnight culture was imaged on
Typhoon 9410 (GE healthcare).

![](https://static.igem.org/mediawiki/2021/3/3b/T--Kyoto--ssrA_004.png)

Left: GFP fluorescence image of each bacterial overnight culture
Right: sequenced three C-terminal amino acids of each culture
The mutant ssrA tag sequence of each clone was sequenced, and biological triplicate
of each bacterial overnight culture was imaged for GFP fluorescence, including controls (LB media
only, bacterial culture not expressing GFP (no GFP), and bacterial culture expressing non
ssrA-tagged GFP (no ssrA tag)).

![](https://static.igem.org/mediawiki/2021/d/d6/T--Kyoto--ssrA_006.png)

The fluorescence of each culture was quantified by ImageJ as inverted mean gray
value. Measured value of each culture was then normalized to the level of the culture expressing non
ssrA-tagged GFP (no ssrA tag), and presented as mean ± SD from three biological replicates. WT (LAA)
and two mutants reported in a previous research are shown in red boxes.

Learn

We mutagenized ssrA tag by PCR with mixed primers, and successfully obtained a mutant
collection of engineered ssrA tag derivatives which show various protein degradation efficiencies.

Cycle5.
 To derive
production efficiency in a production system
using BLOOM

Design

So far, in order to confirm the operation of the production system, the genes of GFP and RFP were used
as the genes to be expressed. Here, in order to discuss the practicality of this system, we decided to
model one of the possible concrete applications of this system.
First of all, we consulted with Ph.D. Ogawa and Ph.D. Takeuchi to get their feedback on how we could
use
this system in a real microbial factory. Then, we got the idea of recovering the bacteria by
agglomerating them and making them bigger, and then searched for a system to realize this.
We decided to use the self-aggregating protein Antigen43, which is known to be expressed on the cell
surface and can aggregate cells to form sediments[4.4][4.5][4.6].

Build

We thought about the system that by incubating *E.coli* introduced BLOOM with continuous
culturing we can
automatically and permanently collect *E.coli* in which the product reached equilibrium, and we
calculated
production efficiency of the system.

Test

We derived the condition that we produce the protein more efficiently by using a model of the 2
plasmid
system. In each *E.coli*, the transition of the concentration of products is shown in the graph
below.

![](https://static.igem.org/mediawiki/2021/c/ce/T--Kyoto--modeling_Modeling_03_01_Prod_prod.png)

Learn

As a result, we found that our system is 5 times more efficient than conventional systems in producing
in 24 hours.

Cycle6.
 Reproduction of 3
plasmid system using BLOOM

Design

In addition to the ParB/ParS pair, it is known that a self-aggregating protein called SopB and a DNA
sequence that binds to SopB called SopC work in a similar manner to cause asymmetric plasmid
partitioning.
Moreover, according to the paper, the parB /parS system and the sopB /sopC system are orthogonal. In
other words, even if the ParB/ParS system and the sopB/sopC system are used at the same time, ParB
will
not be attached to sopC and sopB will not be attached to ParS.
Therefore, we thought that by incorporating sopB/sopC into the above system, we could deepen the
hierarchy of the system and establish a more sophisticated division of roles.

![](https://static.igem.org/mediawiki/2021/8/83/T--Kyoto--Modeling_APP3.png)

1. All cells have both of the three plasmids and are in a stem cell type state. 2 genes, GFP and
   RFP,
   are both repressed by the repressor, but there is a difference in concentration between the two
   repressors due to differences in promoters.
2. When arabinose is added, pBAD is turned on and parB expression is induced.
3. The plasmid with parS aggregates, and at the next division, plasmid asymmetric plasmid
   partitioning occurs, and the plasmid with parS is shed from one of the daughter cells, and it
   becomes differentiated cell I.
4. In differentiated cell I, the concentration of the first repressor decreases because the
   production of new repressors does not occur.
5. When the concentration of the first repressor falls below the threshold for expression
   repression,
   the first gene begins expressing. In this prototype, it is the gene of GFP. In addition to
   it,
   sopB expression is also induced at this time.
6. The plasmid with sopC aggregates, and at the next division, plasmid asymmetric plasmid
   partitioning occurs, and the plasmid with sopC is shed from one daughter cell, and becomes
   differentiated cell II.
7. In differentiated cell II, the concentration of the second repressor decreases because the
   production of new repressors does not occur.
8. When the concentration of the second repressor falls below the threshold for expression
   repression, the second gene begins expressing. In this prototype, it is the gene of RFP.
9. Thus, stepwise gene expression occurs in differentiated cells, and the daughter cells that have
   not shed the plasmid return to (2) or (4), so that the cycle persists semi-permanently.

Build

In order to discuss more complicated systems including APP, we built a model of a 3 plasmid system,
and
simulated the 3 plasmid system.

Test

We succeed in reproducing the phenomenon that two kinds of plasmids are missing in stages as follows.

![](https://static.igem.org/mediawiki/2021/0/03/T--Kyoto--modeling_Modeling_03_02_3pla_copy.png)

Learn

Therefore, we demonstrated that APP can compose more complicated systems, and be widely applicable.

Cycle7.
 Plasmid
construction

Build

Plasmid A acts as a reporter of asymmetric plasmid partitioning. It has iRFP and superfolder GFP. We
used BBa\_K515005 for the superfolder GFP.

![](https://static.igem.org/mediawiki/2021/2/25/T--Kyoto--Engineering_411_plasmidA.png)

Plasmid B is the second self-aggregating plasmid in the three plasmids system. It has lp-ecfp-sopB, cI
repressor, Double terminator, and sopC. We used J23100 for a promoter.

![](https://static.igem.org/mediawiki/2021/4/40/T--Kyoto--Engineering_412_plasmidB.png)

Plasmid C is the first self-aggregating plasmid in the three plasmids system. It has ParS, Double
terminator, TetR, ip-ecfp-parB. We used J23100 for a promoter.

![](https://static.igem.org/mediawiki/2021/e/e4/T--Kyoto--Engineering_413_plasmidC.png)

Plasmid D is self-aggregating in two plasmids system. It has a Double terminator, TetR, cI repressor,
lp-ecfp-parB. We used BBa\_B0015 for a Double terminator. Also, we used J23100 and J23114for promoter.

![](https://static.igem.org/mediawiki/2021/5/58/T--Kyoto--Engineering_414_plasmidD.png)

Test

We constructed plasmids based on the improvement considered in the Learn of cycle1.

Learn

We didn't succeed in constructing plasmids B, C, D. So, we compared the number of colonies and
sequences
derived from self-ligated plasmids.
As a result of the research, it turned out that the problem is due to the characteristics of SLiCE
that
it uses the homology arm to assemble. The principle of SLiCE is almost the same as that of commercial
kits such as InFusion HD. Unexpectedly, the NoTl site sequence common to BioBrick's Prefix and Suffix
assembled as a homology arm. We concluded that these sequences are added to the plasmid to provide
convenience for restriction enzyme cloning. On the contrary, they can be a great obstacle for in vitro
assembly. It is a common problem to many iGEM teams and researchers who construct BioBrick using
InFusion and Gibson Assembly, so we want them to know about it. Moreover, changed the sequence to
attach
primers as shown below, reduced the rate of self-ligated plasmids. When you construct plasmids having
the same Prefix and Suffix, we strongly recommend that using these primers instead of primers
attaching
BioBrick's Prefix and Suffix.

References

4.1 Molinari, S., Shis, D.L., Bhakta, S.P., Chappell, J., Igoshin, O.A., and Bennett, M.R. (2019) "A
synthetic system for asymmetric cell division in Escherichia coli", *Nat. Chem. Biol. 15*,
917–924.
4.2 Kazuhide, S. (2004) (JAPANESE) "確率的 シミュレーションアルゴリズムの時間領域量子化", *BME* Vol.18, No.2
4.3 Flynn, J.M., Levchenko, I., Seidel, M., Wickner, S.H., Sauer, R.T., and Baker, T.A. (2001)
"Overlapping recognition determinants within the ssrA degradation tag allow modulation of
proteolysis",
*Proc. Natl. Acad. Sci. U. S. A. 98*, 10584–10589.
4.4 Ag43+FLAG+MCS http://parts.igem.org/Part:BBa\_K1352001
4.5 Ageorges, V., Schiavone, M., Jubelin, G., Caccia, N., Ruiz, P., Chafsey, I., Bailly, X., Dague,
E.,
Leroy, S., Paxman, J., et al. (2019) "Differential homotypic and heterotypic interactions of antigen
43
(Ag43) variants in autotransporter-mediated bacterial autoaggregation", *Sci. Rep. 9*, 11100.
4.6 https://2012.igem.org/Team:HokkaidoU\_Japan/Project/Aggregation

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# Team:Kyoto/Experiments

[![](https://static.igem.org/mediawiki/2021/2/22/T--Kyoto--images--logoside1.png)](https://2021.igem.org/Team%3AKyoto)

* [Description](https://2021.igem.org/Team%3AKyoto/Description)
* [Implementation](https://2021.igem.org/Team%3AKyoto/Implementation)
* [Proof of Concept](https://2021.igem.org/Team%3AKyoto/Proof_Of_Concept)
* [Engineering](https://2021.igem.org/Team%3AKyoto/Engineering)
* Dry Lab
  + [Modeling](https://2021.igem.org/Team%3AKyoto/Model)
  + [Software](https://2021.igem.org/Team%3AKyoto/Software)
  + [Hardware](https://2021.igem.org/Team%3AKyoto/Hardware)
* Wet Lab
  + [Experiments](https://2021.igem.org/Team%3AKyoto/Experiments)
  + [Results](https://2021.igem.org/Team%3AKyoto/Results)
  + [Parts](https://2021.igem.org/Team%3AKyoto/Parts)
  + [Notebook](https://2021.igem.org/Team%3AKyoto/Notebook)
  + [Safety and Security](https://2021.igem.org/Team%3AKyoto/Safety)
* People
  + [Human Practices](https://2021.igem.org/Team%3AKyoto/Human_Practices)
  + [Education and Communication](https://2021.igem.org/Team%3AKyoto/Communication)
  + [Sustainable Development
    Impact](https://2021.igem.org/Team%3AKyoto/Sustainable)
  + [Excellence in Another Area](https://2021.igem.org/Team%3AKyoto/Gift_from_Synthetic_Biology)
  + [Contribution](https://2021.igem.org/Team%3AKyoto/Contribution)
* Teams
  + [Team](https://2021.igem.org/Team%3AKyoto/Team)
  + [Collaborations](https://2021.igem.org/Team%3AKyoto/Collaborations)
  + [Partnership](https://2021.igem.org/Team%3AKyoto/Partnership)
  + [Attributions](https://2021.igem.org/Team%3AKyoto/Attributions)
* Awards
  + [Result](https://2021.igem.org/Team%3AKyoto/Awards)
  + [Criteria](https://2021.igem.org/Team%3AKyoto/Criteria)

Experiments

Click each title to see detail

1. Virus and Viroid detection

2. Feeding RNAi for western flower thrips

3. Other applications for cut flower maintenance

4. Biomolecular Production Platform

5/30 - 6/5

To conduct our detection assays which would be achieved at less price, we first made homemade enzymes for RT-LAMP, which targets Dahlia mosaic virus (DMV). We transformed pMK715 (for BST DNA polymerase), pMK713, and pMK714 (for Reverse Transcriptase) into competent cells, BL21(DE3), and cultivated them in 1L flasks at 37°C then collected cells and stored them at -80°C until needed.

6/20 - 6/26

We purified the proteins using Ni-NTA beads from the frozen cells. The purification was confirmed by SDS-page.

8/11 - 8/13

The purified protein buffer was replaced with a storage buffer by dialysis to create optimal conditions for the enzymes, followed by the confirmation for their activities.

8/16 - 8/22

To prepare the target sequence for our assay, the part of the DMV genome synthesized by IDT was amplified by PCR. The amplification was confirmed by electrophoresis.

8/30 - 9/5

The activity of BST DNA polymerase we prepared was confirmed by a LAMP assay. Since ladders specific to the LAMP reaction were observed, we concluded that it had activity. After this, we used DNA samples to perform the LAMP assay targeting the DMV sequences. Since amplification plots of the sample using the DNA polymerase we prepared were successfully standing up, just like the plot of positive controls, we confirmed that the LAMP reaction for DMV happened.

9/6 - 9/12

We confirmed the activity of RT(p51 and p66 were coupled) we prepared by a reverse transcription assay using the total RNA of yeasts. When the product after the reverse transcription reaction was electrophoresed after PCR, the band was confirmed, indicating that the RT we prepared was active.

9/26 - 10/2

For detection assay, we prepared crRNA for Cas12a by in vitro transcription. We confirmed the transcription by Qubit.

8/22 - 8/28

Plasmid L4440 (pR005), the vector for dsRNA production, was transformed into DH5a, and the plasmid was collected by miniprep. Using this plasmid, we started the construction of plasmids (pR018, pR019, and pR020) to produce dsRNA for RNAi of Arabidopsis thaliana, Ipomoea nil, and Frankliniella occidentalis. First, we amplified the necessary insert part from gblock with two sets of primers. After that, two fragments of the same insert sequence were inserted in different directions into the restriction enzyme-treated pR005 by INFRONT assembly, and transformed into DH5a. The insert was not amplified due to the duplication of the same sequence, so plasmids from multiple colonies were collected by miniprep and treated with restriction enzymes to confirm the insert length. We found that the target PCR fragment was not inserted from the electrophoresis results, so we gave up the plasmid construction using this method and tried another method. 3 types of gblocks were amplified by PCR, and only VATPase and TrxZ were successfully amplified. We ligated the two successful gblocks into pUC118 which were digested by sma1, which were transformed into DH5α.

8/29 - 9/4

Confirming that the target PCR fragments were inserted in the desired direction by colony PCR, the colonies were cultured and the plasmid was collected by miniprep. Collected plasmids and pR005 were treated with restriction enzymes, electrophoresed, purified by Gel purification. These products were ligated to create plasmids pR015 containing VATPase and pR016 containing TrxZ, and transformed into DH5a.The construction of the target plasmids was confirmed by colony PCR, and the plasmids were collected by miniprep. We retired to amplify EPH1, and inserted it in puc118 using the same protocol. Confirming the construction of target plasmid by colony PCR, the plasmid was collected by miniprep. For additional fragment insertions, six plasmids L4440, pR015, pR016, and pUC118 which have three kinds of fragments were treated by restriction enzymes.

9/5 - 9/11

The second fragments of VATPase and Trxz were inserted in the same method as the first fragment insertion. The first fragment of EPH1 was also inserted.

9/12 - 9/18

The constructed plasmids were collected by miniprep. After treatment with restriction enzymes, the insert lengths were examined by electrophoresis. We successfully constructed all plasmids with one insert (pR015, pR016, 017), while we could not confirm the construction of the plasmids with two inserts. Therefore, we will use the plasmid with one insert to produce RNA.
HT115(DE3) was transformed with pR015, pR017, or pR005 (negative control), and cultured in 1 L of LB medium. RNA was collected by ethanol precipitation and electrophoresed, but the desired band was not detected. Therefore, RNA was purified by additional phenol chloroform treatment and electrophoresed with an acrylamide gel to confirm the production of the RNAs to estimate its concentration. Additional RNA production using 6 L of LB medium to obtain the required amount of RNA for the experiment of Frankliniella occidentalis.

9/19 - 9/25

We collected RNA with phenol chloroform treatment from the additional culture. We also tried to create RNA by in vitro transcription. Each of the RNAs created this week was electrophoresed with an acrylamide gel to confirm the production of RNA.
<Mortality rate assay of Frankliniella occidentalis>
Using dsRNA targeting vATPase-B, an essential gene of Frankliniella occidentalis, prepared by Hiroto Koga and F. occidentalis grown by Dr. Tokumaru of Kyoto Prefectural Agriculture, Forestry and Fisheries Technology Center, we started an RNAi assay to examine the effect of our RNA on the mortality of F. occidentalis. On this day, under the guidance of Dr. Tokumaru, we soaked the petioles of the first green bean leaves in dsRNA solution or Milli-Q. After a few hours, three small leaf fragments were cut out from each green bean leaf, and put with 10 insects in a total of each six petri dishes. The mortality rate will be evaluated after 24, 48, 72, and 96 hours.
<Whitening rate measurement of Arabidopsis thaliana>
We started the RNAi assay to examine the whitening rate of Arabidopsis thaliana using dsRNA targeting the Arabidopsis gene (Trxz) prepared by Hiroto Koga. 200 µl of dsRNA solutions of three different concentrations (0.1, 1, and 10 ng/L) or negative control Milli-Q were added in tubes, and Arabidopsis roots were immersed in each tube. Milli-Q will be periodically replenished and the whitening rate will be measured after one week.

9/26 - 10/2

<Mortality rate assay of Frankliniella occidentalis>
In the insect RNAi assay started last week, the mortality was evaluated every 24 hours until 96 hours later. In particular, the difference in the mortality rate between dsRNA-treated and Milli-Q treated insects 72 hours after the start of the assay was remarkable, with an average mortality rate of 31.3% for dsRNA-treated and 8.3% for Mili-Q treated insects.
<Whitening rate measurement of Arabidopsis thaliana>
One week later, we evaluated the whitening rate of A. thaliana treated with dsRNA solution or Milli-Q last week, but none of them was bleached.
Next, we treated seeds of A. thaliana with a higher concentration (100 ng/µl) of dsRNA and measured the bleaching rate after sprouting. Specifically, we placed a 3 cm square filter paper in the center of each Petri dish, scattered 17 Arabidopsis seeds on the filter paper of the Petri dish, and added 50 µl of 100 ng/µl dsRNA solution or Milli-Q to the paper. We are going to evaluate the bleaching rate when the buds emerge and the leaf length exceeds 5 mm.

8/8 - 8/14

We started to construct plasmids for peptide production: PET11a-CecA-ELK16, PET11a-Def1-ELK16, PET11a-LL37-ELK16, PET11a-NOP1-ELK16 (pR001-pR004). The PCR products were electrophoresed, and column purified. PET11a was cut with restriction enzymes and assembled with the PCR product or the gblock by INFRONT assembly. Assembled plasmids were transformed into DH5a. The transformed E. coli was plated on amp plates and left overnight to form colonies. Considering the possibility of mutation by PCR, we used plasmid assembled with pet11a and gblock. The construction of the target plasmid was confirmed by colony PCR. The selected colonies were cultured in 3 ml of LB for plasmid collection. The plasmid was collected by miniprep and the sequence was confirmed by Sanger sequencing (we would get the result on 8/14). This plasmid was transformed into E. coli BL21(DE3), which was plated on LB amp to produce the peptide.The results of the sequencing were returned on 8/14, and it was confirmed that all four plasmids we prepared had the correct sequences. Based on these results, the construction of four plasmids from pR001-pR004 were completed.

8/15 - 8/21

BL21(DE3) containing each pR001~pR004 were cultured in 1 L of LB medium, and treated with IPTG to induce peptide expression. The cultured E. coli was collected by centrifugation. These were suspended in Buffer and disrupted by French press and sonication. The peptides were precipitated by centrifugation. After washing the precipitates by Buffer repeatedly, they were divided equally, and DTT was added to one of them to cut out peptides by cleavage of inteins for one day. The difficulty of resuspending the peptides and the results of absorbance measurement of the supernatant after washing (especially CecA) suggested the contamination of genomic DNA. Therefore, the other half of the precipitates was sonicated again with two methods to remove the genomic DNA contamination. After this sonication, DTT was added so that only the peptides became soluble. The peptides were collected separately from the precipitates by centrifugation. The production of the peptides was confirmed by Tricine SDS-PAGE. The gel after electrophoresis was fixed.
*<Anti-Bacterial system assay>*
We grew rose, delphinium, and carnation for 10 days without exchanging water. We smeared the vase waters on standard agar medium (peptone 5 g, Glucose 1 g, Yeast extract 2.5 g, and Agar 15 g per 1 L water) by platinum loop and cultured their bacteria for one day. We took each water sample from their stem, the edge of the vase, and water.

8/22 - 8/28

The gel was stained with CBB decolorized. The band of Def1 was confirmed at the target molecular weight, but for the other peptides, major bands at the peptide molecular weight were not confirmed. In addition, there were a lot of impurities in the samples regardless of whether they had been additionally disrupted or not. Therefore, the peptides were purified by ultrafiltration. We did HPLC to confirm that the target peptides were actually contained. From the results of the HPLC, it was found that the peptides contained a lot of impurities when collected as precipitates using ELK16. Therefore, we decided to collect the peptides using His tag instead of ELK16. In order to prepare the plasmids (pR009-pR012) for this purpose, PCR was performed using new primer sets. The plasmids were constructed using the PCR fragments by the same procedure as before, and the plasmids were collected by miniprep. Considering the possibility that the degradation rate changes depending on the amino acid at the N-terminus, we attached valine to the N-terminus of NOP1 and created primers for creating a plasmid for purification with ELK16 (pR013) or His tag (pR014).
*<Anti-Bacterial system assay>*
We again grew rose, delphinium, and carnation for one week. To observe the bacteria, the water in each vase did not exchange. The bacteria were collected on the third day and the fifth day by the same method as before. This experiment showed that the bacteria in the vase changed over time. The bacteria found on the third day (day3 bacteria) seemed to make the environment optimal for the growth of bacteria specific to the fifth day (day5 bacteria). After that, day5 bacteria would change the environment optimal for the next bacteria as well. Therefore, we concluded that to keep the vase water clean, it would be more efficient to reduce bacteria on day3 than day7. We decided to use antimicrobial peptides to kill the day3 bacteria. We used HPLC to confirm CecropinA, Defencin1, LL37, and NOP1 purified by ELK16 and extrafiltered. However, we could not detect their presence.

8/29 - 9/4

The same procedure was performed for pR013 and pR014. After collecting pR013 and pR014, we ordered the sequencing of pR009 to pR012 and pR014. From the result, we found that pR010, 011, 012, and 014 were correctly constructed though the length of the linker between the His tag and the intein varied from plasmid to plasmid due to the codon optimization. We ordered additional sequencing of pR009 and pR013 and confirmed that these plasmids were correctly constructed. The 5 plasmids () for his tag purification or PET11a (negative control) was transformed into BL21(DE3) to form colonies. The single colonies were cultured in 1 L of LB medium and induced the expression of fusion proteins by IPTG, and then the cells were collected by centrifugation. The fusion proteins were extracted and purified using His tag.
*<Anti-Bacterial system assay>*
We cultivated the isolated bacteria from plates on the third and fifth day in liquid medium (peptone 5 g, glucose 1 g, Yeast extract 2.5 g per 1 L water) for one day. After cultivation, we added 300μL of glycerol to 700μL of this liquid culture and kept it at -70℃ as the stock of the bacteria.

9/5 - 9/11

*<Anti-Bacterial system assay>*
We again grew rose, delphinium, and carnation for one week in the same condition as before. To understand the bacterial flora deeply, we took third, fifth- and seventh-day’s water samples for NGS analysis. Taking bacteria from the stock, we cultivated them in a liquid medium for 1 day. After that, we did the blocking circle experiment. We made the standard agar medium that includes one kind of bacteria evenly. The paper discs soaked with antimicrobial peptides were put on that plate and cultured for one day. Finally, we measured the diameter of the inhibition zone.

9/12 - 9/18

The purified proteins were cutted out by adding DTT to get the target peptides. Electrophoresis on Tricine SDS-PAGE was performed to confirm the production of the peptides. The gel after electrophoresis was fixed.

9/19 - 9/25

CBB staining and decolorization of the gel was performed by the same method as before. The band of Def1 was confirmed at the target molecular weight both before and after the addition of DTT. However, for the other peptides, only bands of intein and ELK16 could be seen, and the other peptide bands could not be confirmed clearly.
*<NGS sequencing>*
For bacterial flora analysis, phenol chloroform extraction of metagenomic DNA was performed from the flower vase water samples. Following the DNA extraction, qPCR was performed for total DNA quantification, but the result turned out to be a failure. Next, to prepare the NGS library, the first PCR was conducted on the extracted DNA samples with primer sets containing sample-specific barcode sequences. After the first PCR, the PCR products were column-purified and pooled, followed by the second PCR to fuse sequencing adapter sequences. The PCR products from the second PCR were then size-selected by SPRI paramagnetic beads, and sent to a company for next generation sequencing.
*<Anti-Bacterial system assay>*
We grew rose, delphinium, and carnation for one week in the same condition as before.This time we measured pH of vase water by a pH meter and made plates as well every day. We tried two different ways to culture bacteria. One way is to smear vase water samples by a platinum loop that is the same method as before and the other way is to scatter it on the standard agar medium by a conlarge stick uniformly. In the latter method, we used two different concentration vase water samples. We cultivated Escherichia coli and Bacillus subtilis in a liquid medium for one day and would proceed to the disk diffusion test. Escherichia coli, however, did not multiply, so we used only Bacillus subtilis.
*<NOP-1 assay>*
Pre-experiments were performed in order to consider the conditions of the assay for evaluating the longevity of flowers. In the experiment, flowers of Arabidopsis thaliana grown by Mr. Yuto Ueda and Mr. Hiroto Koga were used and kept under three conditions (grown with tap water, milliQ, or commercial life extension agent).
After that, main-experiments were performed in order to confirm the function of NOP-1(prepared by Mr. Hiroto Koga) and BL・Brz( from Dr. Takeshi Nakano). The flowers used in this experiment were bought at a flower shop in potted form.

9/26 - 10/2

*<NOP-1 assay>*
We took photos and observed flowers everyday. Interestingly, flowers grown with NOP-1 solution lived 3 days longer than those grown with only the buffer. Although the NOP-1 peptide was thought to be NONE because we could not see it in Tricine SDS-PAGE, we may have actually got it. In that case, the reason why we could not see it in Trisin SDS-PAGE was possibly because the NOP-1 peptide was too short. In order to confirm this hypothesis, we asked Mr. Kento Tamukai to make NOP-1 chemically, and we will do the experiments to compare it with the NOP-1 we prepared.

10/3 - 10/9

After ultrafiltration and column purification, mass spectrometry for the correct peptide was performed.
*<Anti-Bacterial system assay>*
We used HPLC to confirm the existence of peptides purified by His tag.
*<NOP-1 assay>*
We bought the flower in potted form at the flower shop again. And we did the same experiment again. This time, we increased the number of samples. In addition to this experiment, we started the experiment with NOP-1 which was made chemically.

8/13 - 8/14

First, we amplified gblocks and biobricks as inserts by PCR. We amplified short inserts by No-template PCR. And we amplified backbone fragments by PCR. We got correct insert fragments but couldn’t get backbone fragments. So, we needed to retry PCR. Also, we needed BBa\_I746916 of iGEM 2021 parts as the PCR template for amplifying the fragment of sfGFP, so it was transformed into DH5a and collected the plasmid by miniprep.

8/15 - 8/21

We retried to amplify the fragments that we had failed to do last week. This time, we used linear plasmids treated by restriction enzymes as a template to amplify the backbone. Prof. Makoto Kitabatake, our PI, gave us a plasmid because SopC is derived from F plasmid. We planned to construct plasmids next week.

8/22 - 8/28

After assembly, we transformed plasmids into DH5a competent cells and incubated them overnight. In plasmid A, red and white colonies were observed, so there was a possibility that DpnI digestion was not sufficient. After colony PCR, we got a band in plasmid B at the targeted size, so we read the sequence.

8/29 - 9/11

This week, we improved the protocol for the construction of plasmids. For example, we considered the conditions for PCR and amplified by overlap extension PCR. But despite these improvements, we could not get the targeted plasmids. Moreover, sequencing revealed a point mutation and a small deletion in the plasmid B sequence.And we had to move to another lab, so we started over the construction of the plasmids. All fragments were amplified by PCR. This time, the backbones were linearized with a restriction enzyme and then amplified by PCR. The PCR products were electrophoresed, and column purified.

9/12 - 9/18

The two fragments were fused by OE-PCR and assembled by Infront assembly. Plasmids A, B, and C were transformed into DH5a and incubated overnight. As a result, a large number of colonies were observed in the negative control plates. From this result, it was considered that the template plasmids remained because column purification was insufficient. So, we decided to purify PCR products by gel purification. Using gel-purified PCR products, we did Infront assembly and transformation again.

9/19 - 9/25

By colony PCR, the bands of plasmid A from two colonies were confirmed at the targeted size, so we sequenced these two samples. As a result, these turned out to be the correct sequences. The reason why other plasmids did not succeed seemed to be the large number of inserts. Therefore, to reduce the number of fragments, we amplified three or more fragments by OE-PCR. In addition, the concentration of parB\_1 and parB\_2 in plasmid B was too low, so we retried PCR. After gel purification, we did Infront assembly and transformation. The result was not successful.

9/26 - 10/2

Based on the results of last week's experiments, we considered the reason why the number of colonies in the negative control was so many. We considered the possibility that the prefix and suffix parts of the backbone sequence were self-ligated. To confirm this hypothesis, we read the sequence of the plasmid gained from the negative control. The results showed that the prefix and suffix parts were self-ligated as we expected, and we needed to redesign the primers. Using the new primers, fragments were amplified by PCR. Since we had all the necessary fragments, we decided to do Infront assembly and transformation again next week.

---

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# Team:Kyoto/Results

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* [Description](https://2021.igem.org/Team%3AKyoto/Description)
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Results

Click each title to switch contents

1. Virus and Viroid detection

2. Feeding RNAi for western flower thrips

3. Other application for cut flower maintenance

4. Biomolecular Production Platform

- Click to visit -

* [Production of homemade enzymes](#sctargetA-1)

+ [Transformation](#sctargetA-1-1)
+ [Protein expression & purification](#sctargetA-1-2)
+ [Dialysis](#sctargetA-1-3)
+ [Checking the activity](#sctargetA-1-4)

* [Cas12a-mediated LAMP/RT-LAMP assay against 2 viruses and a
  viroid](#sctargetA-2)

+ [LAMP against DMV using BST 3.0 and homemade enzyme](#sctargetA-2-1)
+ [Cas12a-mediated LAMP assay against DMV](#sctargetA-2-2)
+ [Cas12a-mediated RT-LAMP assay against CSVd using actual RNA samples from infected plants](#sctargetA-2-3)
+ [Cas12a-mediated RT-LAMP assay against TSWV using actual RNA samples from infected plants](#sctargetA-2-4)

* [References](#sctargetA-5)

Production of homemade enzymes

In order to reduce the cost of one assay of LAMP/RT-LAMP[1.1], we produced homemade DNA polymerase,
and Reverse
Transcriptase. For production, we transformed the plasmids into E.coli, cultured them, and purified
them using Ni-NTA
beads. In a dialysis step we changed the buffers to optimize their activities, and especially for RT,
separately prepared
p66 and p51 were dimerized.

![](https://static.igem.org/mediawiki/2021/0/08/T--Kyoto--Result_101_detection.png)

Transformation

The genes of interest were cloned into pET-11a and were transformed into BL21 (DE3). A lot of colonies
were observed in all the plates. The plasmid maps for each are as follows.
[pMK715](https://benchling.com/s/seq-EdyXpX9J2Ub2IV3KaSrs): DNA polymerase
[pMK713](https://benchling.com/s/seq-zcvAHH6mzGJbiICUwBGU): p66(RT)
[pMK714](https://benchling.com/s/seq-Sgs0tpp1LODLZveCXASN): p51(RT)

Protein expression & purification

Each LB culture was grown at 37 °C to the log-phase. When OD600 reaches 0.35-0.7, we added IPTG at the
final concentration of 0.5 mM. After 3 hours of induction, we cells were harvested and frozen in
liquid-N2. After the cell disruption by sonication, each protein was purified using Ni-NTA beads.
Samples were eluted by a four-step gradient of Imidazole. We observed the correct bands in the
SDS-PAGE gel (shown below). The arrowheads indicate the targeted proteins.

![](https://static.igem.org/mediawiki/2021/f/f5/T--Kyoto--Result_102_detection.png)

Dialysis

We changed the elution Buffer to the storage Buffer for the reaction. We put the purified proteins in
dialysis bags and incubated them o/n at 4 °C in 500 mL of storage buffer. For RT, we added purified p66
and p51 to the same tube and incubated them at -30 °C for 4 days to make a dimer.

Checking the activity

We performed the LAMP reaction and reverse transcription reaction to confirm that the purified
homemade enzymes had activities[1.2][1.3].
*[Checking the activity of homemade DNA polymerase]*
We performed the LAMP reaction using sequence E and its primer set, which already proved to be
successful. We used BST3.0 purchased from NEB as positive control, water as negative controll, and
various gradients of purified homemade BST DNA polymerase as in the following table.

![](https://static.igem.org/mediawiki/2021/2/22/T--Kyoto--Result_103_detection.png)

We performed the LAMP reaction at 63 °C for 45 minutes using a qPCR machine and measured the
fluorescence of Evagreen each cycle. We performed electrophoresis of the samples after the reaction
and observed ladder-like bands ,which is characteristic of the LAMP reaction,in some lanes. From this
result, we confirmed that the homemade DNA polymerase has activity.

![](https://static.igem.org/mediawiki/2021/8/82/T--Kyoto--Result_105_detection.png)

*[Checking the activity of homemade RT]*
In addition to our homemade DNA polymerase, we examined our homemade reverse transcriptase activity in
vitro. We used total yeast DNA/RNA for this assay. We selected the intron-containing region of Rpl19A
as a template for reverse transcription. If there is no reverse transcription, the genomic DNA
including an intron is amplified by PCR, resulting in a band of 876 bp. On the other hand, if cDNA is
correctly synthesized by reverse transcription, a band of 370 bp without an intron should appear.
We carried out reverse transcription experiments under the following conditions, and then PCR.

![](https://static.igem.org/mediawiki/2021/8/80/T--Kyoto--Result_106_detection.png)

As shown below, the cDNA band was successfully observed in an RT-dependent manner. From this result,
we concluded the activity of homemade RT.

![](https://static.igem.org/mediawiki/2021/4/45/T--Kyoto--Result_107_detection.png)

(From left to right: 1-4: newly prepared homemade RT with different enzyme concentrations, 5: no
enzyme, 6: Bst3.0, 7: old homemade RT, 8: no template control)

![](https://static.igem.org/mediawiki/2021/c/c8/T--Kyoto--Result_108_detection.png)

Cas12a-mediated LAMP/RT-LAMP assay against 2 viruses and a viroid

LAMP against DMV using BST 3.0 and homemade enzyme

We performed a LAMP reaction using a part of the DMV genome synthesized by IDT, using both BST3.0 and
a homemade DNA
polymerase[1.4]. From the result of electrophoresis, we confirmed that the LAMP reaction successfully
happened. This
indicates that the primer set is suitable for LAMP.

![](https://static.igem.org/mediawiki/2021/d/d7/T--Kyoto--Result_109_detection.png)

In addition, in the experiment above, isothermal buffer 2 that optimizes the reverse transcription
activity of BST 3.0
was used, but by changing this buffer 2 to buffer 1 that is optimized for DNA amplification, the
activity of DNA
polymerase was increased.

![](https://static.igem.org/mediawiki/2021/3/36/T--Kyoto--Result_110_detection.png)

Cas12a-mediated LAMP assay against DMV

*[Checking that this assay works with BST3.0 and homemade DNA polymerase]*
In order to check that this Cas12a-mediated LAMP assay system works properly, LAMP reaction was
performed with the
following components. These samples were incubated at 63℃ for 50min.

![](https://static.igem.org/mediawiki/2021/0/06/T--Kyoto--Result_111_detection11.png)

After LAMP, we performed Cas12a fluorescent assay in the following procedure and this procedure was
not changed during
all the following experiments.

LAMP reaction at 63℃ for [X]min ( X depends on each experiment. This time, X=50 )
↓
250 nM LbCas12a was first pre-incubated with 250 nM of specific (or non-specific) LbCas12a crRNAs in
1x Cas12 reaction
Buffer for 30 min at 37 ◦C to assemble Cas12-crRNA ribonucleoprotein (RNP) complexes.
↓
The reaction was diluted 4 times with a NEB 1x binding buffer.
↓
For fluorescence-based detection,
for Cas12a 250 nM and for Cas12b, 500 nM of fluorophore-quencher (FQ) reporter (5′-/56-FAM/TTATT/
3IABKFQ/-3′, IDT)
and 2 µl of the RT-LAMP reaction was added to the pre-assembled Cas12- sgRNA RNP complexes and
incubated at 37 ◦C for
10–30 minbr

After the assay, we observed the fluorescence of FAM with the naked eye under a blue light reader.
Then, we measured
FAM values quantitatively using a qPCR machine. With both BST3.0 and the homemade DNA polymerase, we
were able to detect
about 10-15 fold fluorescence than the negative control. This confirms that the Cas12a-mediated LAMP
assay works both
with BST3.0 and with the homemade BST DNA polymerse.

![](https://static.igem.org/mediawiki/2021/9/97/T--Kyoto--Result_113_detection.png)

*[Examination of the detection limit of the Cas12a-mediated LAMP assay against DMV]*
In order to verify the sensitivity of the Cas12a-mediated LAMP assay, we performed Cas12a-mediated
LAMP assay with
different copy numbers of DMV target DNA, from 10^10 copies to 1 copy, under the following conditions.
LAMP was
conducted at 63 °C for 30 min.

![](https://static.igem.org/mediawiki/2021/0/07/T--Kyoto--Result_014_detection.png)

After the assay, we checked the fluorescence of FAM with the naked eye under a blue light reader.
Since there was no
significant difference between these 12 wells, we also measured the value of FAM quantitatively by a
qPCR machine. From
the following graphs, we concluded that BST3.0 can detect up to one copy.

![](https://static.igem.org/mediawiki/2021/2/24/T--Kyoto--Result_115_detection.png)

*[Verify that assay works with BST2.0 warm start]*
The purchased enzyme BST3.0 which we had been using was out of stock, so we switched to using BST2.0
warmstart as an
alternative enzyme. We confirmed the function of this enzyme by the same protocol as before. However,
the amount of the
enzyme was changed from 1 unit to 8 units as recommended by NEB[1.5].

![](https://static.igem.org/mediawiki/2021/e/ef/T--Kyoto--Result_116_detection.png)

After the assay, we checked the fluorescence of FAM with the naked eye under a blue light reader.
Then, we also measured
the value of FAM quantitatively by a qPCR machine. However, as shown in the following graph, we only
observed the
detection of 10^10copies. Therefore, we changed the amount of dNTP and MgSO4 of mix, but we could not
raise the
detection limit at all.

![](https://static.igem.org/mediawiki/2021/0/03/T--Kyoto--Result_117_detection.png)

Cas12a-mediated RT-LAMP assay against CSVd using actual RNA samples
from infected
plants

Since the Cas12a-mediated LAMP assay that detects the synthetic DNA sequence was working well, we
proceeded to the
Cas12a-mediated RT-LAMP assay to also detect RNA viruses.
In these experiments((3) and (4)), we aimed to detect CSVd and TSWV using RNA samples obtained from
actual infected
plants. We got these RNA samples from a collaborator. He kindly gave us two RNA samples of each.
Since BST polymerase 2.0 has reverse transcription activity, we used twice the amount of the enzyme to
perform the RT-
LAMP reaction. Since we were told to dilute the samples 20 times, we prepared a 3-step gradient of
samples: original
concentration, diluted 20 times, and a no-template one. For RT-LAMP reaction, samples in the following
table were
incubated at 63 °C for 30 minutes.

![](https://static.igem.org/mediawiki/2021/2/20/T--Kyoto--Result_118_detecton.png)

After the assay, we checked the fluorescence of FAM with the naked eye under a blue light reader. We
also measured the
value of FAM quantitatively by a qPCR machine, but as shown in the graph, sufficient fluorescence
could not be confirmed.

![](https://static.igem.org/mediawiki/2021/2/25/T--Kyoto--Result_119_detection.png)

Cas12a-mediated RT-LAMP assay against TSWV using actual RNA samples
from infected plants

We also performed Cas12a-mediated RT-LAMP assay on TSWV samples[1.6]. For TSWV we prepared two types
of
crRNA, and we performed the RT-LAMP reaction by incubating the samples in the following table at 63 °C
for 30 min.

![](https://static.igem.org/mediawiki/2021/0/05/T--Kyoto--Result_120_detection.png)

After the assay, we checked the fluorescence of FAM with the naked eye under a blue light reader. We
also measured the value of FAM quantitatively by a qPCR machine, but as shown in the graph, sufficient
fluorescence could not be confirmed this time ,either. The problems seem to be that the quality of the
RNA samples was not confirmed and the optimal conditions for BST2.0 warm start were not sufficiently
examined, so we would like to solve these in the future.

![](https://static.igem.org/mediawiki/2021/8/8e/T--Kyoto--Result_121_detection.png)

References

1.1 Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N., and Hase, T.
(2000) "Loop-mediated
isothermal amplification of DNA", *Nucleic Acids Res. 28*, E63.
1.2 Ali, Z., Aman, R., Mahas, A., Rao, G.S., Tehseen, M., Marsic, T., Salunke, R., Subudhi, A.K.,
Hala, S.M., Hamdan,
S.M., et al. (2020). iSCAN: An RT-LAMP-coupled CRISPR-Cas12 module for rapid, sensitive detection of
SARS-CoV-2. *Virus
Res. 288*, 198129.
1.3 Seckler, J.M., Howard, K.J., Barkley, M.D., and Wintrode, P.L. (2009) "Solution structural
dynamics of HIV-1 reverse
transcriptase heterodimer", *Biochemistry 48*, 7646–7655.
1.4 Lee, S.H., Baek, Y.H., Kim, Y.-H., Choi, Y.-K., Song, M.-S., and Ahn, J.-Y. (2016) "One-Pot
Reverse Transcriptional
Loop-Mediated Isothermal Amplification (RT-LAMP) for Detecting MERS-CoV", *Front. Microbiol. 7*,
2166.
1.5 *New England Biolabs*, "Loop-Mediated Isothermal Amplification", https://international.neb.com/applications/dna-amplification-pcr-and-qpcr/isothermal-amplification/loop-mediated-isothermal-amplification-lamp
1.6 Fukuta, S., Ohishi, K., Yoshida, K., Mizukami, Y., Ishida, A., and Kanbe, M. (2004) "Development
of immunocapture
reverse transcription loop-mediated isothermal amplification for the detection of tomato spotted wilt
virus from
chrysanthemum", *J. Virol. Methods 121*, 49–55.

* [dsRNA production](#sctargetB-1)

+ [Design](#sctargetB-1-1)
+ [Plasmid construction](#sctargetB-1-2)
+ [RNA production](#sctargetB-1-3)

* [Thrips assay](#sctargetB-2)

+ [Overview](#sctargetB-2-1)
+ [Assay method and results](#sctargetB-2-2)

* [Determination of whitening rate in Arabidopsis](#sctargetB-3)

+ [Overview](#sctargetB-3-1)
+ [Assay method and results](#sctargetB-3-2)

* [References](#sctargetB-4)

dsRNA production

Design

We conducted this experiment with the aim to silence the target gene by inducing RNAi by ds RNA
produced in E. coli. The target genes for this experiment are an essential gene of Frankliniella
occidentalis, a gene responsible for chloroplast formation during early development in Arabidopsis
thaliana, and a gene for a transcription factor that positively regulates PCD in Ipomoea nil.
Silencing these genes is expected to kill the Frankliniella occidentalis, whitening leaf of
Arabidopsis thaliana, and double the flowering time of Ipomoea nil [2.1][2.2][2.3].
In producing dsRNA in E. coli, the plasmid design was based on the following paper [2.4][2.5]. A
plasmid containing the two target gene sequence assembled in different orientations in the region
between the two T7 promoters transformed into E. coli strain HT115 (DE3), which has low RNase activity
and expresses T7 polymerase. Transformed E. coli transcribe hairpin RNA.
In addition, the following paper was used as a reference for the purification of dsRNA from E. coli
culture[2.6].

Plasmid construction

Plasmid construction 1

Connect the L4440 which is treated by restriction enzyme with a part of the target gene by INFRONT
assembly.
As a result,the plasmid was not created correctly. It is thought that the plasmid left over when the
vector was treated with restriction enzymes appeared as a colony. Therefore, we gave up on this method
and tried a new method.

![](https://static.igem.org/mediawiki/2021/4/48/T--Kyoto--Engineering_104_Plasmid_Design.png)

Plasmid construction 2

Multiple restriction enzymes were combined to create the target plasmid.

![](https://static.igem.org/mediawiki/2021/5/5d/T--Kyoto--Engineering_103.1_DNAgel2.jpg)

(1insertion 1, 2 VATPase-B 3,4 Trxz-3 5,6 EPH1
2insertion 7,8,9 Trxz-3 10,11,12 VATPase-B )

I was able to confirm that the insertion of one fragment was successful. For 7~12, a band of about 1100
bp is a correct insertion of two inserts. c shows a fragment of the correct length, but other than
that, no bands of the correct length can be seen.
Therefore, 7~12 is examined by cutting it with another enzyme (Xho1).
If the correct insert had been inserted, two cuts would have occurred and two bands would have been
visible. However, since only one band can be seen as a major band, it is thought that the second
insert was not inserted correctly.

![](https://static.igem.org/mediawiki/2021/0/04/T--Kyoto--Result_201_RNAproduction.png)

(2insertion 1,2,3 Trx-Z 4,5,6 V-ATPase )

The three plasmids with one insert (pR015, pR016, and pR017) are considered complete, and the plasmid
with two inserts (pR019) is considered provisionally complete.

RNA Production

HT115 (DE3) is made into competent cells by calcium chloride method and transformed with plasmids
(pR005, pR015, pR016, pR017, pR019).

![](https://static.igem.org/mediawiki/2021/3/3e/T--Kyoto--Result_202_RNAproduction.png)

Enough colonies could be confirmed.
Considering the priority of the experiment, HT115 transformed with pR015 was cultured in two 1L LB,
one each with pR017 and pR005.
The culture was started at 37 °C and IPTG was added when OD exceeded 0.4 to induce RNA production for 4
hours[2.7]. E. coli was disrupted and precipitated with NaCl, and RNA contained in the supernatant was
collected by ethanol precipitation. Based on the results of absorbance measurement of the recovered
RNA, it was assumed that many impurities other than the target RNA were contained in the RNA solution.

![](https://static.igem.org/mediawiki/2021/3/3b/T--Kyoto--Result_205_RNA.png)

Collected RNA by ethanol precipitation after phenol-chloroform treatment.
Electrophoresis in acrylamide gel was performed to confirm whether the target RNA was produced.

![](https://static.igem.org/mediawiki/2021/9/98/T--Kyoto--Engineering_105_RNAgel1.jpg)

(1: VATPase1, 2: VATPase2, 3: EPH1, 4: L4440 5: VATPase1, 6: VATPase2, 7: EPH1, 8: L4440)

There is a major band around 700 bp（▲）, which is considered to be the target product because the band
visible in EPH1 is a little shorter than that of vATPase-B.
The yield was estimated based on the results of this electrophoresis.
Additional production of dsRNA for vATPase-B was also performed in 6L LB medium. The production method
was the same protocol as the previous one. For recovery of dsRNA, since phenol chloroform treatment
was found to be necessary in the previous production, E. coli was crushed with NaCl and then purified
by phenol chloroform treatment.

![](https://static.igem.org/mediawiki/2021/3/37/T--Kyoto--Engineering_105_1_rnagel2.jpg)

1.L4440 (add production) 1ug 2 vATPase-B add production (7ug) 3 vATPase-B(add
production)（1ug）4 vATPase-B-1 (1ug）5 vATPase-B-2 (1ug), in vitro RNA production（6 vATPase-B,7 Trxz,8
EPH1）1ug each)

We were able to see a band that could be considered as the target dsRNA in this preparation as well as
the previous one. In addition, a band of similar length was also observed in the in vitro
transcription system.
The production amount of dsRNA was examined based on the electrophoresis result.

![](https://static.igem.org/mediawiki/2021/e/eb/T--Kyoto--Engineering_106_Table_rna.jpg)

In the case of production by E. coli, the yield was much higher the second time when the recovery
method was simplified. There is a possibility that the amount lost by ethanol precipitation is much
higher than expected. In addition, the accuracy of the results is questionable because some of the
expected concentrations have changed significantly from the last time they were electrophoresed.
Compared to the in vitro transcription system, the production in E. coli is considered to be more cost
effective.

Thrips assay

Overview

Insect pests are one of the major problems in the production stage of the flower industry. In
particular, western flower thrips feed on a variety of flowers such as roses and carnations, and have
been selected as one of “The 100 most invasive alien species” in Japan. In addition, it transmits
Tomato spotted wilt virus (TSWV), a virus that has become a serious problem in production along with
pests.
We have demonstrated that feeding dsRNA targeting essential genes of western flower thrips can induce
RNAi and kill the pest. The dsRNA used in this assay was produced by E. coli.

Assay method and results

Specifically, 200 µl of a 200 ng/µl dsRNA solution targeting vATPase-B (Milli-Q was used as a negative
control) was pipetted in a PCR tube and the petiole of a green bean primary leaf was
immersed[3.1][3.2][3.3]. After 2
hours, three leaf fragments of 1 cm × 2 cm were cut from the soaked bean leaves and placed in three
petri dishes with about 10 thrips. The mortality and leaf damage were checked after 24, 48, 72, and 96
hours.
The results are shown below.

![](https://static.igem.org/mediawiki/2021/d/da/T--Kyoto--File_Proof_009_RNAi2.png)

In particular, there was a large difference in the mean mortality rate between dsRNA-treated and
negative control thrips on day 3: 8.3% and 31.3%, respectively. This result confirms the insecticidal
effect of dsRNA indirectly feeding to the western flower thrips.

Determination of whitening rate in Arabidopsis

Overview

Endogenous genes in plants play a major role in flower senescence. If the genes involved in floral
senescence can be knocked down using external dsRNA, it is expected that floral senescence will be
suppressed and flowers will last longer.
We used a model plant, Arabidopsis thaliana, to see if RNAi could be induced by dsRNA solutions
targeting an important endogenous gene. The dsRNA used in this assay was synthesized by in vitro
transcription.

Assay method and results

Specifically, 200 µl of 0.1, 1, or 10 ng/l dsRNA solution targeting Trxz (Milli-Q was used as a
negative control) was placed in a PCR tube, and Arabidopsis roots were immersed. For this assay, Dr.
Shimada provided us with Arabidopsis plants that had sprouted after being sown on the plate on August
31.
The condition of Arabidopsis after one week is shown below.

![](https://static.igem.org/mediawiki/2021/7/73/T--Kyoto--Result_203_RNA.png)

From the left lane, negative control, Arabidopsis soaked in 0.1, 1, and 10
ng/L dsRNA solution (three replicates)

![](https://static.igem.org/mediawiki/2021/0/09/T--Kyoto--Result_204_RNA.png)

From the left lane, negative control, Arabidopsis soaked in 0.1, 1, and 10
ng/L dsRNA solution (three replicates)

As a result, the RNAi effect was not well observed as a whole. In this study, the RNAi assay was
performed by calculating the concentration of dsRNA obtained by in vitro transcription from the values
measured by NanoDrop. However, electrophoresis showed that only a small amount of dsRNA in the target
band was included. Therefore, the low concentration of dsRNA is
considered to be the reason why the RNAi effect was not seen well.

References

2.1 Andongma, A.A., Greig, C., Dyson, P.J., Flynn, N., and Whitten, M.M.A. (2020) "Optimization of
dietary RNA interference
delivery to western flower thrips Frankliniella occidentalis and onion thrips Thrips tabaci",
*Arch. Insect Biochem. Physiol.
103*, e21645.
2.2 Chen, Z., He, J., Luo, P., Li, X., and Gao, Y. (2018). Production of functional double-stranded
RNA using a prokaryotic
expression system in Escherichia coli. Microbiologyopen e787.
2.3 Shibuya, K., Shimizu, K., Niki, T., and Ichimura, K. (2014) "Identification of a NAC
transcription factor, EPHEMERAL1,
that controls petal senescence in Japanese morning glory", *Plant J. 79*, 1044–1051.
2.4 Tenllado, F., Martínez-García, B., Vargas, M., and Díaz-Ruíz, J.R. (2003) "Crude extracts of
bacterially expressed dsRNA
can be used to protect plants against virus infections", *BMC Biotechnol. 3*, 3.
2.5 Timmons, L., and Fire, A. (1998) "Specific interference by ingested dsRNA", *Nature 395*,
854.
2.6 https://2019.igem.org/Team:SZU-China/Experiment.
2.7 Papić, L., Rivas, J., Toledo, S., and Romero, J. (2018) "Double-stranded RNA production and the
kinetics of recombinant
Escherichia coli HT115 in fed-batch culture", *Biotechnol Rep (Amst) 20*, e00292.
3.1 Andongma, A.A., Greig, C., Dyson, P.J., Flynn, N., and Whitten, M.M.A. (2020) "Optimization of
dietary RNA interference
delivery to western flower thrips Frankliniella occidentalis and onion thrips Thrips tabaci.
*Arch. Insect Biochem. Physiol.
103*, e21645.
3.2 Han, S.H., Kim, J.H., Kim, K., and Lee, S.H. (2019) "Selection of lethal genes for ingestion RNA
interference against
western flower thrips, Frankliniella occidentalis, via leaf disc-mediated dsRNA delivery. Pestic",
*Biochem. Physiol.
161*, 47–53.
3.3 Singh, S., Gupta, M., Pandher, S., Kaur, G., Goel, N., Rathore, P., and Palli, S.R. (2019) "RNA
sequencing, selection of
reference genes and demonstration of feeding RNAi in Thrips tabaci (Lind.) (Thysanoptera:
Thripidae)", *BMC Mol. Biol. 20*,
6.

- Click to visit -

* [Peptide production](#sctargetC-1)

+ [Design](#sctargetC-1-1)
+ [Peptide production](#sctargetC-1-2)
+ [New plasmid construction](#sctargetC-1-3)
+ [N end rule](#sctargetC-1-4)
+ [Second peptide production](#sctargetC-1-5)
+ [MS](#sctargetC-1-6)

* [Anti-Bacterial system assay](#sctargetC-2)

+ [Overview](#sctargetC-2-1)
+ [the observation of the bacterial flora of the vase water](#sctargetC-2-2)
+ [Disk Diffusion method](#sctargetC-2-3)

* [NOP-1 assay](#sctargetC-3)

+ [Assay 1](#sctargetC-3-1)
+ [Assay 2](#sctargetC-3-2)

* [Biofilm degrading enzyme experiment](#sctargetC-4)

+ [Overview](#sctargetC-4-1)
+ [Assay](#sctargetC-4-2)

* [References](#sctargetC-5)

Peptide production

Design

The following experiments were conducted to reduce the number of bacteria that cause vessel blockage
and prolong the life of flowers by producing antibacterial peptides in E. coli and adding them to
vases.
We also produced and added a peptide called NOP1, which inhibits ethylene signaling, in the same way
to prolong the life of cut flowers from a different perspective. The following paper was used as a reference to produce the peptide[4.1][4.2][4.3].
The peptide is expressed as a fusion protein: peptide-Mxe
GyrA
intein-linker-ELK16. The fusion protein aggregates and precipitates due to the action of ELK16. When
DTT is added to the precipitated fusion protein, self-cleavage occurs at the N-terminus of the intein,
and the peptide is cut out into the water layer. To produce the fusion protein, a plasmid was created
by assembling the gblock encoding the fusion protein and pet-11a. The following three types of
antimicrobial peptides were used: Defensin-1, Cecropin-A, LL37[4.4][4.5][4.6].

Peptide production

Plasmid construction

As shown in the figure, PET11a was cut with restriction enzymes (BamH1, Nde1), and gblocks coding the
sequences of fusion protein connected by INFRONT assembly. The plasmid is completed as designed.

![](https://static.igem.org/mediawiki/2021/c/cc/T--kyoto--plasmid_ELK16.png)

Peptide production

The production of peptides was carried out with reference to[4.3].
The plasmids from pR001 to pR004 were transformed into E. coli BL21 and colonies were formed on the
plate.
Each colony was transferred to 1L LB medium for culture and production of peptide.
The culture was started at 37 °C. The OD was measured sometimes, and when the OD exceeded 0.35, IPTG
was used to induce peptide expression. E. coli was collected as a precipitate after incubation.

![](https://static.igem.org/mediawiki/2021/0/09/T--Kyoto--Result_301_peptideproduction.png)

Def1 looked a little reddish, unlike the other precipitations. This may be the color of the expressed
Def1.
E. coli collected as precipitate was suspended in BufferB1 (20 mM TrisHCl 8.5, 500 mM NaCl, 1 mM EDTA)
and disrupted by French pressing twice and sonication (10 sonicate, 1 min) six times.
The precipitate containing the fusion protein was collected by centrifugation.
The precipitates were washed by suspending them in Buffer B1, centrifuging them, and discarding the
supernatant five times to remove the soluble components in the precipitates. The precipitates were
divided equally, and Buffer B3 (20 mM TrisHCl 8.5, 500 mM NaCl, 1 mM EDTA 40 mM DTT) was added to one
of the precipitates, and peptides were cut out by cleavage of inteins for one whole day. The other
half of the collected precipitates were sonicated through water (2s sonication, 2s rest) x 100 or
normal sonication (2s sonication, 2s rest) x 100 for additional disruption. The precipitates were then
washed by suspending them in BufferB1, centrifuging and discarding the supernatant, repeated three
times. After washing, the precipitates were mixed with buffer B3 to separate the peptides from the
precipitates.

Electrophoresis on Tricine SDSPAGE

In order to confirm the actual existence of the peptides, we performed electrophoresis with Tricine
SDSPAGE. Tricine SDSPAGE can detect proteins with smaller molecular weight than normal SDSPAGE. The
results of the electrophoresis were as follows.

![](https://static.igem.org/mediawiki/2021/5/57/T--Kyoto--Engineering_203_SDS-PAGE%E2%91%A0_kai.png)

The band that is considered to be correct in Def1 (5713 Da) appears, while it could not be confirmed as a major
band in the rest of the peptides. Also, the mass of the fusion protein of intein and ELK16 is about 25
kDa, and this band is also visible. It seems that the impurities have been reduced by additional
sonication, but still many impurities remained.
An ultrafiltration was performed to remove impurities above 10 kDa for HPLC and assays.

New plasmid construction

There are three problems that we found during the first round of peptide purification. First, when the
fusion protein is recovered as a precipitate using ELK16, the purified peptide contains many
impurities
even after washing.Second, the precipitates contain highly viscous material such as genomic DNA,
which
makes it difficult to repeat peptide production because of the long time required to wash the
precipitates. Third, peptides other than Def1 cannot be identified as major bands, and the amount
recovered is quite low. As a second peptide production strategy to solve these problems, we used a
system that uses His tag and intein. The peptide-containing fusion protein can be recovered in a
column
by using the binding of His tag to nickel beads.

![](https://static.igem.org/mediawiki/2021/4/47/T--Kyoto--purasumido_His.png)

Using an additional primer, a DNA fragment of His tag-intein-peptide was amplified by PCR from the
gblock of ELK16-intein-peptide. INFRONT assembly was done to create a plasmid.

N end rule

The effect of n-end-rule was also considered as the cause of the problem of low recovery[4.7].
About E. coli, the following rules exist
Proteins which have Arginine, lysine, leucine, phenylalanine, tyrosine, and tryptophan at the amino
terminal have a half-life of two minutes. On the other hand, other peptides have a half-life of more
than 10 hour.
In the plasmid we created, only Def1 has a long half-life, while CecA, LL37, and NOP1 have short
half-lives.
I thought that this might have been the reason for the low recovery of peptides other than Def1, so we
designed to improve the N-terminal of the peptides. Create two plasmids, one for Histag purification
and
one for ELK16 purification, for the production of NOP1 peptides with a Valine at the N-terminal
end.

Second peptide production

Plasmid construction

PCR was used to create a DNA fragment of fusion protein include NOP1v.INFRONT assembly was done to
create a plasmid.
In plasmid construction, pR009 to pR014 were completed.

Peptide production

The plasmid for His tag purification and the control pet11a were transformed into BL21. From the
colonies on the plate, we cultured them in 1L of LB medium at 37C. After IPTG induction, the culture
was
transferred to 30C for 6 hours and the E. coli that produced the peptide was collected as a
precipitate.
The cultured E. coli was crushed by sonication and washed with bufferA. The target protein was eluted
with Ni-NTA beads at four concentrations of Imidazole: 70 mM, 95 mM, 120 mM, and 270 mM.

![](https://static.igem.org/mediawiki/2021/7/7b/T--Kyoto--File_peptide_result_999_SDS.png)

peptide (concentration of Imidazole)
1.NOP1v(270 mM) 2.NOP1v(120mM) 3.NOP1(270mM) 4.NOP1(120mM) 5.Def1(270mM) 6.Def1(120mM)
7.CecA(270 mM)
8.CecA(120 mM) 9. LL37(270 mM) 10.LL37(120 mM)11.Pet11a(270 mM) 12.Pet11a(120 mM)

Purification of proteins using the His-tag was more successful than using ELK16
After purification of fusion protein, add DTT for these to cut out peptides.

![](https://static.igem.org/mediawiki/2021/5/53/T--Kyoto--Engineering_204_SDS-PAGE%E2%91%A1.pptx_%282%29.png)

(1 CecA 2 Def1 3 LL37 4 NOP1 5 NOP1v 6 pet11a 7 CecA 8 Def1 9 LL37 10 NOP1 11 NOP1v)

The peptides were electrophoresed by Tricine SDS PAGE, and the bands of the peptides could be seen in
Def1 with and without DTT. In all lanes except pet11a, a band (of 24 kDa) that seems to be HIs-intein
is
visible. Therefore, the expression of the fusion protein and the purification of the fusion protein
using His tag seems to have been successful. There are three possible reasons why the other peptides
were not identified as bands. First, NOP1 is a particularly short peptide and may have been run away
during electrophoresis. The second possibility is that the peptides were not cleaved by the
self-cleavage of the intein, and the third possibility is that the intein was cleaved at some point
before purification and moved to another fraction during imidazole elution. Def1 was also cut out
before

MS

Mass spectrometry was performed by Bruker Daltonics flexAnalysis. 0.5 µl of sample was mixed with 0.5 µl
of Matrix solution and air-dried. After that, the plate was set and calibrated by standard and
insulin,
and the measurement was started. The results are shown below. The possible molecular weights are shown
in
the table below.

PDF ( click to expand PDF viewer )

However, we did not observe the expected molecular weights for all peptides, which is questionable
since
the Tricine SDS PAGE data suggests that the fusion proteins themselves were produced. It is possible
that the excised peptides have undergone unnecessary modification or cleavage in the cell, or that
they
have been partially degraded.

Anti-Bacterial system assay

Overview

One of the major causes of wilting cut flowers is that bacteria growing in the vase invades the vessel
and prevents flowers from absorbing water. Solving this problem, we decided to use antimicrobial
peptides from organisms to inhibit growth of them. Although there are many kinds of antimicrobial
peptides, not all of them are capable of attacking all species of bacteria. Considering that, we tried
to analyze what kinds of bacteria existed in the vase and use some antimicrobial peptides to kill them
effectively.
To show that, we had grown flowers for one week. We took water from each sample and spread it on the
standard agar medium（Agar 15g Glucose1g peptone 6g Yeast extarct2.5g per 1L）in order to observe the
changes in bacterial flora. Besides, we did the disk diffusion in order to see if the antimicrobial
peptides we purified could actually inhibit the growth of bacteria.
*KeyAchievement :*
We found that the bacterial flora of the vase had changed as the days went by.
The antimicrobial peptides we purified did not show the antimicrobial activity.

The observation of the bacterial flora of the vase water

Material and method

We choose carnation and rose that are famous for and delphinium that the florist recommends as a
flower
that trends to cloud the water. We had grown them for one week without changing their water. We took
50 µl water from each of the stem, the vase surface, and water for that one week and spread it on the
standard agar medium by conlarge stick. When we smeared water samples, we diluted them as there were
about 1000 colonies according to each OD600. Also, this one week, we measured pH. The water was
absorbed
by flowers or evaporated, so we added water and kept 30mL for one week.

![](https://static.igem.org/mediawiki/2021/3/3a/T--Kyoto--Result_321_antibacteria.png)

We did the same thing twice as a preliminary experiment. From the results of them, the species of
bacteria became similar after about one week. So, in this experiment, I decided to observe them for a
week.

Changes in water in the vase over time

Fig.1 shows the results of the normal tap water changes in bacterial flora. As it shows, the tap water
from day1 to day4 could not grow any bacteria, but from day5 to day7, many kinds of bacteria rapidly
increased.

![](https://static.igem.org/mediawiki/2021/3/30/T--Kyoto--Result_322_antibacteria.png)

Fig.1-Changes in pH and bacterial flora of normal tap water

![](https://static.igem.org/mediawiki/2021/3/32/T--Kyoto--Result_323_antibacteria.png)

Fig.2-OD600 and dilution rate of normal tap water

Changes in bacterial flora focusing on each flower or the place

From Fig.3, as for rose, the red bacteria appeared on the day4 in the vase water. After that, the
green one became dominant. Taking into account the pH change, it increased on day4 with an increase of
red one and decreased on day5 with an increase of green one.

![](https://static.igem.org/mediawiki/2021/e/e4/T--Kyoto--Result_324_antibacteria.png)

Fig.3-Changes in bacterial flora and pH of rose

![](https://static.igem.org/mediawiki/2021/7/7d/T--Kyoto--Result_325_antibacteria.png)

Fig.4-OD600 and dilution rate of rose

Although the bacterial amount in the vase of carnation was so large compared to other flowers, the clear bacterial change would not show. The similar bacteria appeared in the stem and the side of vase. Besides, the bacteria in carnation would grow earlier than two.

![](https://static.igem.org/mediawiki/2021/6/64/T--Kyoto--Result_326_antibacteria.png)

Fig.5-Changes in bacterial flora and pH of carnation

![](https://static.igem.org/mediawiki/2021/7/74/T--Kyoto--Result_327_antibacteria.png)

Fig.6-OD600 and dilution rate of carnation

As for delphinium, the red bacteria appeared at the same time pH increased. After that, when the yellow one was found, pH decreased.

![](https://static.igem.org/mediawiki/2021/7/75/T--Kyoto--Result_328_antibacteria.png)

Fig.7-Changes in bacterial flora and pH of delphinium

![](https://static.igem.org/mediawiki/2021/1/10/T--Kyoto--Result_329_antibacteria.png)

Fig.8-OD600 and dilution rate of carnation

In this experiment, in both rose and delphinium, the red bacteria that we had not seen before appeared
on the day4, but on the day5, they disappeared. We did not know if these two red bacteria were the
same. However, we think that what is important would be that the bacterial flora in the vase between
on the early day and the later was different.
We think that the three factors would cause this trend. First, the many kinds of bacteria in the air
could fall into the vase that we set up. In this experiment, we did not put the vase in the sterile
condition to recreate the situation of growing flowers at home. So, the bacteria or dust from the air
could accumulate in the vase and change its bacterial flora.
Second, although it is thought that this cause might be more influential, the secretion from the
flower could make the vase water more nutrient, which changed the environment in the vase. Although
there were no bacteria in the early vase water, on the day6 or 7, the water had been so dirty you
could see it. The degree or rate of contamination varied by species, which could show that the
composition of vase water could rely on the kind or amount of secretion from flowers. This would
change the water continuously and lead to the variation of bacteria that adapt and grow in each water
environment.
Third, a more interesting possibility is that environmental changes that the previous bacteria had
caused prevented the growth of the bacteria that was previously the dominant one[5.3]. Such changes in
dominant species, which have been well studied in the case of plankton in natural lakes, are observed
in a variety of cases. It is thought that this is partly due to environmental changes as a result of
the flourishing of dominant species, such as the depletion and decomposition of their preferred
nutrients and the accumulation of waste products that hinder their growth. In this experiment, we also
noted that the pH of the water increased at the same time as the red colony bacteria increased. There
is no evidence that the red colony is directly responsible for this pH change. However, we observed
that at least one environmental factor changed during the change of the dominant species.
From these experiments we have learned that the flora of the water in the vase is dynamically
changing. This led us hypothesize that in order to suppress the environmental changes in the vase
water in advance and to make flowers live longer consequently, it would be more effective to use
antimicrobial substances to the bacterium in relatively clean water on the second or third day of the
experiment than to use them to the bacterium in dirty water on the seventh day.
In this experiment, we also examined the flora of samples collected from different parts of the vase.
The results showed that the bacterium did not grow on stems or the vase surface first, but rather
moved from the water to the stems and vase surface. This suggests that antibacterial substances should
be added to the water first, before they are added to the stems and vases. It might be an effective
way to investigate the initial bacterium appearing in the water and to use the antibacterial
substances to them.

Disk Diffusion method

![](https://static.igem.org/mediawiki/2021/a/ac/T--Kyoto--Result_330_antibacteria.png)

The result of the disk diffusion method

Using the peptides synthesized and collected in this project, we tested their antimicrobial activity
by disk diffusion test[5.4]. On the previous day, the bacteria were incubated in liquid medium
(Glucose 1g,
peptone 6g, Yeast extract 2.5g per 1L water) at 300rpm for one day at 28℃ with shaking.
Standard agar medium (Agar 15g, Glucose 1g, peptone 6g, Yeast extract 2.5g per 1L water) was
autoclaved. After waiting until the medium becomes 50 °C, 50 µl of bacteria solution was added per 20mL
of standard agar medium to make plates with bacteria.
Paper discs were soaked in 50 µl of antimicrobial peptide and placed on a plate with bacterium and
cultivated at 27 °C for one day to see if an inhibition zone was observed.
First, the disk diffusion method was performed with peptides generated by the ELK16system.
We used the bacteria from the water of the flowers on the 3 and 5 day. However, in this experiment,
they did not show the antimicrobial activity. Later, the same experiment was carried out using
peptides purified by His tag. In this experiment, we used Bacillus subtilis as the indicator bacteria,
because in the previous experiment, we might have accidentally selected a bacteria that was resistant
to the antimicrobial peptide. As a result, we could not observe the antimicrobial activity. (In the
picture below, we used CecA, Def1 and LL37 purified by His tag.)
Ampicillin (10,500,1000 µg/ml) was used as positive control.

NOP-1 assay

Assay 1

Materials and Methods

To research the effect of NOP-1 and NOP-1v synthesized by E.coli, we conducted this assay. We used
potted Dianthus that has an ethylene-dependent pathway.
We didn’t use cut flowers to avoid prior treatment with STS. Following previous research, We used 1mM
AgNO3 aq as Positive control and the same buffer for NOP-1 as Negative control[6.1]. Also, We
confirmed the
effect of BL and Brz that Professor Nakano gave us[6.2]. We used the same buffer for BL or Brz as
Negative
control.

The Result of Assay 1

Note that the start date of this assay should be the 1st day.
*The effect on life prolongation byNOP-1 and NOP-1v synthesized by E.coli*

![](https://static.igem.org/mediawiki/2021/6/6b/T--Kyoto--Result_351_NOP1_assay1.png)

![](https://static.igem.org/mediawiki/2021/a/a4/T--Kyoto--Result_352_NOP1assay.png)

As shown above, NOP-1 and NOP-1v extend the flower life by 1-2 days against the Negative control. And
it seemed to have the same life prolongation as 1mM AgNO3 used as the positive control.
*The effect on life prolongation by BL and Brz Professor that Nakano gave us*

![](https://static.igem.org/mediawiki/2021/d/db/T--Kyoto--Result_353_NOP1assay.png)

![](https://static.igem.org/mediawiki/2021/1/15/T--Kyoto--Result_354_NOP1assay.png)

As shown above, Brz extended the life of flowers for two days against the negative control. And it
seemed to have the same life prolongation as 1mM AgNO3 used as the positive control. In addition, BL
did not have the same life prolongation as Brz. These results were similar to the findings of
Professor Nakano, who gave us BL and Brz.
NOP-1, NOP-1v, and Brz seemed to have life prolongation of Dianthus. However, the number of each
sample was only one. So we were not able to conclude the effects, and we retried to conduct an
assay.

Assay 2

Materials and Methods

To confirm the result of assay 1, we increased the number of samples and retried an assay. We also
researched the effect of chemical synthesized NOP-1. We used the same buffer for NOP-1 as the Negative
control.

The Result of Assay 2

Note that the start date of this assay should be the 1st day.
*the effect on long prolongation by NOP-1 and NOP-1v synthesized by E.coli*

![](https://static.igem.org/mediawiki/2021/8/89/T--Kyoto--Result_355_NOP1assay.png)

*the effect on long prolongation by chemical synthesized NOP-1*

![](https://static.igem.org/mediawiki/2021/5/5b/T--Kyoto--Result_356_NOP1assay.png)

Both NOP-1 and NOP-1v in vivo and NOP-1 in vitro didn't show life prolongation.

Biofilm degrading enzyme experiment

Overview

We had thought of antimicrobial peptides as the main countermeasure against bacteria in vases.
However, after consulting with Dr. Nomura, we learned that even in a vase, bacteria are likely to form
biofilms to protect themselves from antimicrobials.
Therefore, we decided to investigate the effect of biofilm-degrading enzymes on flowers by
collaborating with the iGEM team of Gunma University, with whom we had formed a partnership.
*Key achievements :* The ability of the enzyme could not be confirmed.

Assay

Preparing dahlias and asters

We went to Hanachu florist near Kyoto University, and bought flowers based on the diameter of their
stems: thick and thin. We chose dahlias and asters as a sample. Each of them was put in a vase filled
with 200 mL of tap water and left them at room temperature for three days.

Check the activity of BF enzyme

After 3 days, flowers were removed from the vase and cut into 1cm, 2cm, and 3cm pieces from the tip.
Each flower was cut into two along the diameter of the face and one was used as the control group and
the other as the experimental group.
Put the stems into a 1.5ml tube, add PBS, and shake it. Centrifuge and remove the PBS with an
aspirator. This was done a total of three times (3000 rpm). Then the control group was treated with
200 µl of PBS, and the study group was treated with 200 µl of Buffer + 10 µl of enzyme. After adding
the reagent, it was incubated overnight at 37℃.
Enzyme samples from Gunma Univ. require a dedicated buffer. Prepared the reagent listed below and PBS
was added to become 10mL.

![](https://static.igem.org/mediawiki/2021/d/dc/T--Kyoto--Result_360_biofilm.png)

qPCR

Nucleic acids were extracted from the bacteria by processing the samples with lysozyme and Protein K
incubated overnight. Nucleic acids were purified by phenol and chloroform, and the DNA retrieved by
ethanol precipitation was subjected to qPCR. The genomic DNA of DH5alpha, which was measured as a
standard, was amplified and a quantitative line was generated. The results are shown below. However,
we could not obtain any data for the sample because it was below the detection limit.
This may be due to the fact that the activity of the enzyme was weaker than expected and the fungus
did not fall off from the stem, or it fell off when washed in PBS. It is also possible that the BF
produced on the flowers could not be degraded by the α-galactosidase of Gunma University.

![](https://static.igem.org/mediawiki/2021/0/0b/T--Kyoto--Result_361_biofilm.png)

qPCR for genomic DNA of DH5alpha

References

4.1 Hoppen, C., Müller, L., Albrecht, A.C., and Groth, G. (2019) "The NOP-1 peptide derived from
the central regulator of
ethylene signaling EIN2 delays floral senescence in cut flowers", *Sci. Rep. 9*, 1287.
4.2 Wang, M., Zheng, K., Lin, J., Huang, M., Ma, Y., Li, S., Luo, X., and Wang, J. (2018) "Rapid
and efficient production of
cecropin A antibacterial peptide in Escherichia coli by fusion with a self-aggregating protein",
*BMC Biotechnol. 18*, 62.

4.3 Lin, Z., Zhao, Q., Zhou, B., Xing, L., and Xu, W. (2015). Cleavable Self-Aggregating Tags
(cSAT) for Protein Expression and
Purification. In Insoluble Proteins: Methods and Protocols, E. García-Fruitós, ed. (New York, NY:
Springer New York), pp. 65–
78.
4.4 Part:BBa K1230001 - parts.igem.org. http://parts.igem.org/Part:BBa\_K1230001
4.5 Part:BBa K1104301 - parts.igem.org. http://parts.igem.org/Part:BBa\_K1104301
4.6 Cecropin A. https://www.medchemexpress.com/Cecropin\_A.html?locale=ja-JP
4.7 Tobias, J.W., Shrader, T.E., Rocap, G., and Varshavsky, A. (1991) "The N-end rule in bacteria",
*Science 254*, 1374–
1377.
5.1 Florack, D.E.A., Stiekema, W.J., and Bosch, D. (1996) "Toxicity of peptides to bacteria
present in the vase water of cut
roses", *Postharvest Biol. Technol. 8*, 285–291.
5.2 Mancinelli, R.L., and Shulls, W.A. (1978) "Airborne bacteria in an urban environment", *Appl.
Environ. Microbiol. 35*,
1095–1101.
5.3 Simek, K., Pernthaler, J., Weinbauer, M.G., Hornák, K., Dolan, J.R., Nedoma, J., Masín, M.,
and Amann, R. (2001) "Changes
in bacterial community composition and dynamics and viral mortality rates associated with enhanced
flagellate grazing in a
mesoeutrophic reservoir", *Appl. Environ. Microbiol. 67*, 2723–2733.
5.4 Tenover, F.C. (2009). Antibiotic Susceptibility Testing. In Encyclopedia of Microbiology
(Third Edition), M. Schaechter,
ed. (Oxford: Academic Press), pp. 67–77.
6.1 Hoppen, C., Müller, L., Albrecht, A.C., and Groth, G. (2019) "The NOP-1 peptide derived from
the central regulator of
ethylene signaling EIN2 delays floral senescence in cut flowers", *Sci. Rep. 9*, 1287.
6.2 Nakagawa, Y., Nishikawa, B., and Miyagawa, H. (2021) "Effects of brassinolide on the growing
of rice plants", *J. Pestic.
Sci. 46*, 274–277.

* [ssrA tag mutagenesis](#sctargetD-1)

+ [Key achievements](#sctargetD-1-1)
+ [Plasmid construction](#sctargetD-1-2)
+ [Imaging bacterial overnight culture under blue light](#sctargetD-1-3)
+ [Sequences and GFP intensity of mutant ssrA tags](#sctargetD-1-4)

* [Plasmid construction](#sctargetD-2)

+ [Design](#sctargetD-2-1)
+ [Construction](#sctargetD-2-2)

* [References](#sctargetD-3)

ssrA tag mutagenesis

Please also refer to Engineering Success for the design: [Engineering Success](https://2021.igem.org/Team%3AKyoto/Engineering)

Key achievements

We mutagenized ssrA tag by PCR with mixed primers, and successfully obtained a mutant collection
of
engineered ssrA tag derivatives which show various protein degradation efficiencies.

Plasmid construction

First, we tried to obtain the backbone and insert fragments needed for the plasmid
construction.
Backbone fragment was PCR-amplified from pSB4K5-J04450.

![](https://static.igem.org/mediawiki/2021/9/98/T--Kyoto--ssrA_010.png)

The size of the desired band is 3584bp. Each PCR reaction was checked on agarose gel, and the
one
amplified from 1.0ng of the template was chosen and column-purified because of low
background.
Insert fragment was obtained by 2 steps of PCR.
The first PCR was performed on the GFP insert derived from BBa\_K584001 to add the first 24-bp
sequence
of ssrA tag, which is not the target for mutagenesis.

![](https://static.igem.org/mediawiki/2021/0/00/T--Kyoto--ssrA_009.png)

The size of the desired band is 798bp. Each PCR reaction was checked on agarose gel, and the one
amplified from 0.01ng of the template was chosen.
The first PCR product was then diluted 1:1000 and used as the template for the second PCR to
fuse and
mutagenize the 9-bp target sequence for mutagenesis with mixed primers.The primers LAA, AAV,
ASV, and
XXX were used individually, but XAA, LXA, and LAX were pooled and used in the same reaction.

![](https://static.igem.org/mediawiki/2021/4/45/T--Kyoto--ssrA_008.png)

The size of the desired band is 798bp. Each PCR reaction was checked on agarose gel and
column-purified.
The column-purified backbone and insert fragments were assembled by InFront Assembly (a homology
arm-mediated seamless cloning method like In-Fusion), and transformed into E.coli competent
cells.

Imaging bacterial overnight culture under blue light

As a result of transformation, colonies of various fluorescence intensity were obtained on the
LB
plates.. 72 colonies in total were picked from plates, and cultured overnight in LB media. The
image
of E.coli cultures were taken under the blue light.

![](https://static.igem.org/mediawiki/2021/2/22/T--Kyoto--ssrA_007.png)

![](https://static.igem.org/mediawiki/2021/2/26/T--Kyoto--ssrA_011.png)

A10: LB media only
B10: E.coli culture not expressing GFP
B7: E.coli culture expressing non ssrA-tagged GFP
C7: E.coli culture expressing WT ssrA-tagged GFP

Bacterial cultures of various GFP intensity were observed.

Sequences and GFP intensity of mutant ssrA tags

Plasmid was extracted from each bacterial culture by miniprep, and then Sanger-sequenced to
identify ssrA tag sequence. Furthermore, to quantify protein degradation efficiency of each
mutant, GFP Fluorescence of each bacterial overnight culture was imaged on Typhoon 9410 (GE
healthcare).

![](https://static.igem.org/mediawiki/2021/3/3b/T--Kyoto--ssrA_004.png)

The mutant ssrA tag sequence of each clone was sequenced, and biological triplicate of each
bacterial overnight culture was imaged for GFP fluorescence, including controls ( LB media only,
bacterial culture not expressing GFP (no GFP), and bacterial culture expressing non ssrA-tagged
GFP (no ssrA tag)).

![](https://static.igem.org/mediawiki/2021/d/d6/T--Kyoto--ssrA_006.png)

The fluorescence of each culture was quantified by ImageJ as inverted mean gray value. Measured
value of each culture was then normalized to the level of the culture expressing non ssrA-tagged
GFP (no ssrA tag), and presented as mean ± SD from three biological replicates. WT (LAA) and two
mutants reported in a previous research are shown in red boxes.

Plasmid construction

Design

To produce biomaterials more efficiently and environmentally sustainable, we adopted a system of
cell differentiation and division of roles named “asymmetric plasmid partitioning." We
constructed plasmid A required for this system.
Plasmid A has iRFP and superfolder GFP. We used BBa\_K515005 for the superfolder GFP controlled
by cI repressor and TetR, this plasmid act as a reporter of asymmetric plasmid partitioning.

Construction

We amplified inserts and backbone fragments by PCR.

![](https://static.igem.org/mediawiki/2021/d/d7/T--Kyoto--result_production_kai11.png)

lane 1: pSB1A3-mRFP. The band looked correct (2155bp).

![](https://static.igem.org/mediawiki/2021/7/7d/T--Kyoto--result_production_kai2.png)

lane 1 and 6: iRFP. The bands looked correct. (1009bp)

![](https://static.igem.org/mediawiki/2021/0/0c/T--Kyoto--result_production_kai32.png)

lane 1: oligo\_A. The bands looked correct. (137bp)

![](https://static.igem.org/mediawiki/2021/b/bd/T--Kyoto--result_production_kai41.png)

lane 1: superfolder GFP. The band looked correct. (789bp)

All PCR products looked correct, so we purified them with columns. All dsDNA concentration was
enough
(pSB1A3 : 93.2 ng/ul, iRFP : 44.2 ng/ul, oligo\_A : 44.2 ng/ul, superfolder GFP : 99.4 ng/ul). To
reduce the
number of inserts, we fused inserts by OE-PCR.

![](https://static.igem.org/mediawiki/2021/0/0a/T--Kyoto--result_production_kai51.png)

lane 1: OE-PCR1 (iRFP and oligo\_A, 1116bp), lane 2: OE-PCR2 (oligo\_A and superfolder GFP, 896bp)

Both of them look correct. We purified OE-PCR1 because its band looks clearer. dsDNA
concentration of
that was 155.6 ng/ul. We got all fragments for plasmid A, so we assembled them (mistakenly we
used iRFP
instead of superfold GFP). After that, we transformed them to DH5a and incubated them overnight.
The
plates after incubation are shown below.

![](https://static.igem.org/mediawiki/2021/d/d1/T--Kyoto--Result_416_production_conctruct6.png)

From the left to the right, plasmid A (×1/10), plasmid A (×1), plasmid
A control (only iRFP and pSB1A3 fragments)

Too many bacteria were cultured, so we were not able to separate each colony. This cause may
inactivate ampicillin, or there was some contamination. To solve these failures, we purified
them by
gel purification. After gel purification, the dsDNA concentration of pSB1A3 and superfolder GFP
were
enough. (pSB1A3 : 12.1 ng/uL, superfolder GFP : 27.6 ng/uL) But that of OE-PCR1 was too low. So
after
this, we purified fragments only by gel purification. We retried, dsDNA concentration of the
dsDNA
concentration of RFP was enough. (15.9 ng/uL) So, we retried assembly and transformation. The
plates
after incubation are shown below.

![](https://static.igem.org/mediawiki/2021/b/b6/T--Kyoto--Result_417_production_conctruct7.png)

from the left to the right, plasmid A (×1/10), plasmid A (×1),
plasmid A control (only superfold GFP
and pSB1A3 fragments).

We picked eight colonies each from plasmid A and A control and amplified them by colony PCR. We
used
two sets of primers, one set covering all of the inserts and the other covering a part of the inserts.

![](https://static.igem.org/mediawiki/2021/b/bb/T--Kyoto--result_production_kai6.png)

lane 1-8: plasmid A (primer: covering all of the inserts), lane 9-16: plasmid A control (primer: covering all of the inserts), lane 17-24: plasmid A (a part of the inserts)

![](https://static.igem.org/mediawiki/2021/f/f3/T--Kyoto--result_production_kai72.png)

lane 25-32: plasmid A control (a part of the inserts)

1, 3 lanes looked correct (2146 bp). And 17, 19 lanes looked correct (1620 bp), so we read the
sequence of them. After the miniprep, the concentrations of plasmid were 109.3 ng/µl and 139.3
ng/ul. As a result of the reading sequence, both of the plasmids have the correct sequence.So,
we succeeded to construct plasmid A.

References

7.1 Djurhuus, A., Port, J., Closek, C.J., Yamahara, K.M., Romero-Maraccini, O., Walz, K.R.,
Goldsmith, D.B., Michisaki, R.,
Breitbart, M., Boehm, A.B., et al. (2017) "Evaluation of Filtration and DNA Extraction Methods
for Environmental DNA
Biodiversity Assessments across Multiple Trophic Levels", *Frontiers in Marine Science 4*,
314.
7.2 Flynn, J.M., Levchenko, I., Seidel, M., Wickner, S.H., Sauer, R.T., and Baker, T.A. (2001)
"Overlapping recognition
determinants within the ssrA degradation tag allow modulation of proteolysis", *Proc. Natl.
Acad. Sci. U. S. A. 98*,
10584–10589.

---

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