iGEM 2020 — Team NYMU-Taipei

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# Team:NYMU-Taipei/Notebook

* [Home](https://2020.igem.org/Team%3ANYMU-Taipei)
* Team

  [Members](https://2020.igem.org/Team%3ANYMU-Taipei/Team)
  [Attributions](https://2020.igem.org/Team%3ANYMU-Taipei/Attributions)
* Project

  [Description](https://2020.igem.org/Team%3ANYMU-Taipei/Description)
  [Design](https://2020.igem.org/Team%3ANYMU-Taipei/Design)
  [Results](https://2020.igem.org/Team%3ANYMU-Taipei/Results)
  [Model](https://2020.igem.org/Team%3ANYMU-Taipei/Model)
  [Software](https://2020.igem.org/Team%3ANYMU-Taipei/Software)
  [Hardware](https://2020.igem.org/Team%3ANYMU-Taipei/Hardware)
  [Implementation](https://2020.igem.org/Team%3ANYMU-Taipei/Implementation)
  [Contribution](https://2020.igem.org/Team%3ANYMU-Taipei/Contribution)
* Parts

  [Parts](https://2020.igem.org/Team%3ANYMU-Taipei/Parts)
  [Engineering](https://2020.igem.org/Team%3ANYMU-Taipei/Engineering)
* [Safety](https://2020.igem.org/Team%3ANYMU-Taipei/Safety)
* HP

  [Human Practices](https://2020.igem.org/Team%3ANYMU-Taipei/Human_Practices)
  [Collaborations](https://2020.igem.org/Team%3ANYMU-Taipei/Collaborations)
  [Education](https://2020.igem.org/Team%3ANYMU-Taipei/Education)
  [Partnership](https://2020.igem.org/Team%3ANYMU-Taipei/Partnership)
* Interlab

  [Notebook](https://2020.igem.org/Team%3ANYMU-Taipei/Notebook)
  [Experiments](https://2020.igem.org/Team%3ANYMU-Taipei/Experiments)
* Awards

  [Awards](https://2020.igem.org/Team%3ANYMU-Taipei/Awards)
  [Criteria](https://2020.igem.org/Team%3ANYMU-Taipei/Criteria)

## Notebook

![First slide](https://static.igem.org/mediawiki/2020/3/31/T--NYMU-Taipei--notebook_display_1.jpg)

![Second slide](https://static.igem.org/mediawiki/2020/9/9b/T--NYMU-Taipei--notebook_display_2.jpg)

![Second slide](https://static.igem.org/mediawiki/2020/f/f8/T--NYMU-Taipei--notebook_display_3.jpg)

[Previous](#team-photos)
[Next](#team-photos)

## May - Brainstorming and topic decision

* We looked back to numerous igem teams project in the past few years and
  tried to choose our topic. We then reached a consensus to do a project that will
  help
  improve the situation of COVID-19, which is ravaging the world. After tons of
  discussion
  and document reading, we planned to fight the threatening corona virus by targeting
  the
  spike protein on it’s surface.

## June - Gain more information and construct our project

* We came up with several ideas to avoid being infected by SARS-CoV2,
  such
  as FRET assay for detection, using a protease for inactivation, and some
  applications
  that may help our design. Divided into small groups, we started to look deep into
  our
  plan and design every part in detail. Lots of paper research are done in this phase
  and
  an initial prototype of our project is formed.

## 6/29-7/5 - First week in the lab

* Lab doings

  We did a comprehensive clean up of our lab, including sterilizing
  containers we might use, throwing away expired solutions from the fridge and
  putting on new bench paper.
  Made account and collected virus genome data from database.
* Other issues

  We started to look for the sequences we may need to purchase form
  addgene and learn how to design primers for later plasmid construction.

## 7/6-7/12 - The start of wet lab

* Lab doings

  We made some LB plates with different antibiotics and started to
  get familiar with the instruments in the lab
* Other issues

  We listed the plasmid we may construct and decide which method to
  connect the DNA fragments (Restriction enzyme ligation or Gibson assembly)

## 7/13-7/19

* Lab doings

  We did LB plate planting and plasmid extraction to the bacteria
  with plasmids we bought from addgene, then use restriction enzymes to check if
  we got the right plasmid. We also made a list of what restriction enzymes and
  DNA markers we have in the lab.
  Made preliminary results from virus sequence analysis.
* Other issues

  We communicated with NCKU igem team and are looking forward to
  have further discussion and exchanges later in the summer.

## 7/20-7/26

* Lab doings

  Continued to do plasmid extraction and store them in the fridge
  for later PCR.
  Modeled virus protein structural sequence to see important mutation sites in 3D.
* Other issues

  We had a vigorous discussion about which kit and what tag(SUMO or
  his tag) we are going to use for the protein purification of our fusion protein
  constructs. Also, we decided what linker to use for our fusion protein after a
  series of paper research.

## 7/27-8/2

* Lab doings

  We cut the plasmid (Scarlet I-FRET 10-Venus) with restriction
  enzymes and then collected the sequence of Venus fp and Scarlet-I fp by gel
  electrophoresis and gel extraction. The results of gel extraction is known by
  nanodrop spectroscopy.
* Other issues

  We spent a great afternoon having an interview with doctor Zhan
  and learnt a lot from his experience and advices.

## 8/3-8/9

* Lab doings

  Keep doing plasmid extraction (because some of the plasmids remain
  having low concentration) and restriction enzyme digestion as last week.
* Other issues

  Some of us visited a professor in Chung Xing University to get
  information about the use of chitin tag and how to utilize the chitin tag to let
  our construct bind to the contact lens’ surface.
  However, The professor advised us not to use chitin tag and introduced another
  method using merely his tag to achieve our goal.

## 8/10-8/16

* Lab doings

  PCR progress: Venus FP, ACE2, bacterial expression vector
* Other issues

  We started to construct our wiki using hackMD website. The rough
  framework of our wiki is formed.

## 8/17-8/23

* Lab doings

  PCR progress: Venus and Scarlet FP, ACE2
  Gibson assembly progress: ACE2 RBD
* Other issues

  We had an online seminar with NCKU igem team, at which exchange
  our thoughts and discuss about the challenges we met and find a solution. We
  also discussed about our collaboration about human practice related to contact
  lens (which is a part we have in common).

## 8/24-8/30

* Lab doings

  PCR progress: pepN(failed), spike protein(low yield)
  Transformation: ACE2( to BL21 strain).
* Other issues

  We had another online seminar with CSMU igem team and had a good
  time sharing our thoughts and advices about each other’s’ project. We also had a
  lab collaboration(PCR double check) scheduled.

## 8/31-9/6

* Lab doings

  PCR progress: pepN(failed), spike protein(low yield)
  Transformation: ACE2( to BL21 strain)
* Other issues

  We visited Prevision contact lens factory and demonstrated our
  design to them.

## 9/7-9/13

* Lab doings

  PCR progress: pepN(failed), spike protein(low yield)
* Other issues

  We started our collaboration events with NCKU igem team,
  propagating citizens not to discard contact lens carelessly after use.(Slogan:
  Throw contact lens properly, marines life live more happily) Also, we started to
  draw up the framework of our team promotion video.

## 9/14-9/20

* Lab doings

  PCR progress: pepN(failed), spike protein(low yield).
* Other issues

  We find out that making contact lens as our only product may be
  lacking persuasion, so we discussed if there are other kinds of application our
  construct can be used.
  Also, the protease we chosen (pepN) was found not able to cut spike protein, so
  we immediately initiated a new search for a replacement.

## 9/21-9/27

* Lab doings

  PCR progress: ACE2, Venus & Scarlet FP, Spike protein(low yield).
* Other issues

  We’ve finalized our team promotion video and determined our
  project name “Off The Crown”. We also decided which track of igem we are
  participating in.
  We came up with another protease(pepP) ordered the primers to construct its
  plasmid. Furthermore, in case the spike protein is too big for the protease to
  cut, we extracted the four pepP cleavage sites on spike protein and created
  fragments that has 35bp of DNA front and behind the cleavage site.

## 9/28-10/4

* Lab doings

  We’ve finalized our team promotion video and determined our
  project name “Off The Crown”. We also decided which track of igem we are
  participating in.
  We came up with another protease(pepP) ordered the primers to construct its
  plasmid. Furthermore, in case the spike protein is too big for the protease to
  cut, we extracted the four pepP cleavage sites on spike protein and created
  fragments that has 35bp of DNA front and behind the cleavage site..
* Other issues

  The second plan of our application is determined (detection box DeBux), and we
  started to design the structure of the box and preparing to use 3D printing to
  form its structure.

## 10/5-10/11

* Lab doings

  Started to construct the gel we are going to stick our fusion
  protein to(Using poly HEMA).
  Gibson assembly progress: pepP only(success)
  Transformation: pepP only(to BL21 strain).
* Other issues

  Wiki description page and home page design finished.
  Criteria “excellence in another area” done (collaboration with NCKU igem team).
  Integrated human practice recorded and organized.

## 10/12-10/18

* Lab doings

  PCR progress: cut 1-4 spike fragment & vector, P+A pepP, ACE2, &
  vector
  Gibson assembly progress: spike cut 3&4
  Protein purification: pepP, RFP, ACE2
  Perform oxidization to our gel(KMnO4).
* Other issues

  Start writing our judging form.

## 10/19-10/25

* Lab doings

  Gibson assembly progress: spike cut 1-3, PepP+ACE2(success)
  Protein purification: pepP, RFP, Spike cut4, ACE2
  Test the ability of our gel to bind to his tagged protein.
  pepP cleavage test(use SDS PAGE and Western Blot to check the results.
* Other issues

## 10/26-10/28

* Lab doings

  SDS-Page: spike cut 1-4, pepP, RFP
  Protein purification: pepP, RFP, Spike cut 1-4
  Make hydrogel and oxidize the gel.
  Test the ability of our gel to bind to GFP with his tag.
* Other issues

  Complete our wiki.

A Project conducted by the 2020 NYMU iGem Team

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nymuigem2020@gmail.com

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@wetlabtenpeaple

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NYMU iGEM Team @igemnymutaipei

Copyright 2020 NYMU iGem Team. All Rights Reserved

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# Team:NYMU-Taipei/Engineering

* [Home](https://2020.igem.org/Team%3ANYMU-Taipei)
* Team

  [Members](https://2020.igem.org/Team%3ANYMU-Taipei/Team)
  [Attributions](https://2020.igem.org/Team%3ANYMU-Taipei/Attributions)
* Project

  [Description](https://2020.igem.org/Team%3ANYMU-Taipei/Description)
  [Design](https://2020.igem.org/Team%3ANYMU-Taipei/Design)
  [Results](https://2020.igem.org/Team%3ANYMU-Taipei/Results)
  [Model](https://2020.igem.org/Team%3ANYMU-Taipei/Model)
  [Software](https://2020.igem.org/Team%3ANYMU-Taipei/Software)
  [Hardware](https://2020.igem.org/Team%3ANYMU-Taipei/Hardware)
  [Implementation](https://2020.igem.org/Team%3ANYMU-Taipei/Implementation)
  [Contribution](https://2020.igem.org/Team%3ANYMU-Taipei/Contribution)
* Parts

  [Parts](https://2020.igem.org/Team%3ANYMU-Taipei/Parts)
  [Engineering](https://2020.igem.org/Team%3ANYMU-Taipei/Engineering)
* [Safety](https://2020.igem.org/Team%3ANYMU-Taipei/Safety)
* HP

  [Human Practices](https://2020.igem.org/Team%3ANYMU-Taipei/Human_Practices)
  [Collaborations](https://2020.igem.org/Team%3ANYMU-Taipei/Collaborations)
  [Education](https://2020.igem.org/Team%3ANYMU-Taipei/Education)
  [Partnership](https://2020.igem.org/Team%3ANYMU-Taipei/Partnership)
* Interlab

  [Notebook](https://2020.igem.org/Team%3ANYMU-Taipei/Notebook)
  [Experiments](https://2020.igem.org/Team%3ANYMU-Taipei/Experiments)
* Awards

  [Awards](https://2020.igem.org/Team%3ANYMU-Taipei/Awards)
  [Criteria](https://2020.igem.org/Team%3ANYMU-Taipei/Criteria)

* [Phase 1](#Phase1)
* [Phase 2](#Phase2)
* [Phase 3](#Phase3)

### Engineering

---

The very start goal of our project is to eliminate the suspending COVID-19 virus
particles in the air. Through synthetic biology, we could achieve specific capture
and inactivation. This could be helpful in highly dangerous or intricate working
place, such as the custom or IC chip factory, since the air flow needs extra
protection.

---

#### Phase One Design (March to July)

Through researching previous experiments, we found out that huamn receptor hACE2.
This could be used to capture the flowting virus in the air to fulfill our need.
Next, in order to inactivate the virus, we cameup with an idea to malfunction the
virus with a protease. According to documentation, COVID-19 virus enter human cell
through the binding between spike protein and human receptor hACE2. After that, a
human membrane protein called the TMPRSS2 will cut spike protein on S1/S2 domain,
enable the virus to enter human cell through membrane funsion.

Thus, our phase one design is to construct a fusion protein, which is consist of
hACE2, a linker and a selected protease. The protease is choosing according to
peptidase database “MEROPS”. We wish to find something only reacts with spike
protein rather than hACE2. The protease we choose was aminopeptidase N(pepN), which
is a E. coli strain K12 MG1655 protein.

As for how to detect the binding of spike protein and hACE2, we planned to apply a
FRET design between the two proteins. In our design, we choosed to put both
fluorescent proteins at the N terminal of spike and ACE2. Therefore, when the spike
protein and hACE2 bind to each other, the acceptor of FRET pair will emit light due
to the decrease of distance between the FRET pair.

---

#### Phase Two Design (Summer Vacation)

During experiment, we found various problems about our original design. First, the
fusion protein with Fret(hACE2+linker+protease+fret) is far too big to construct and
express in E. coli(over 5000 bp). Second, the cloning of pepN wasn’t too easy, since
the genomic DNA of E. coli horbors more than 4M bp gene sequence. We had quite a
hard time adjusting the PCR condition. Lastly, there were still cleavage sites on
hACE2, which could lead to the inactivation of out fusion protein.

To solve these problems, we later add a his tag on the end of the plasmid to replace
the usage of Fret protein. The result will be demonstrate through western blotting
gel. The mass of protein various before and after protease cleavage. We also adjust
the denaturing time in the three-step cycle of PCR from standard 30 second to 3
minutes, which is meant to fully open the 4M genomic DNA. Lastly, we choose to use
hACE2’s RBD(receptor-binding domain) rather than full length in order to eliminate
possible cleavage site on it.

What’s noteworthy is that we choose KOD as our PCR enzyme in order to prevent
mutation cause by DNA polymerization. In comparision t enzyme Taq, KOD has a better
proofreading accuracy and thermostability. This could provide a better quality for
our engineering product. Moreover, Gidson assemble was used in order to combine
multiple DNA fragments simultaneously, enhancing the efficiency of our experiment.

---

#### Phase Three Design(September ~)

In the final pahse of our experiment, we replaced pepN with pepP after analyzing the
structure of pepN. This protease is structurally enclosed, making it hard to react
with big protein substrate such as spike protein. PepP(aminopeptidase P) is another
protease existing in E. coli strain K12 MG1655. The structure of pepP is a C-like
bar, which is relatively open and likely to react to larger substrate.

According to MEROPS’s data, PepP has four cleavage sites on spike protein(326 ~ 333
IVRFPNIT, 487 ~ 494 NCYFPLQS, 1049 ~ 1056 LMSFPQSA, 1086 ~ 1093 KAHFPREG, between
F&P). However, pepP left a cleavage site on hACE2’s RBD(protein sequence 364~371
DMAYAAQP, between Y&A), which could lead to further malfunction problem. As a
result, we created a point mutation on hACE2, changing sequence YAA into AAA to
prevent protease reaction. This design ensures the fusion protein intact and
function normally.

Finally, in case pepP can’t react with full length spike protein, we created spike
fragments of the cleavage sites. As research pointed out, pepP can cut small
fragments of protein(roughly 4.15~4.2 kDa). Each fragment are attached with his tag.
With western blotting method, we could differentiate different protein after
reaction.

---

##### References

1. Lan, J., Ge, J., Yu, J. et al. Structure of the SARS-CoV-2 spike receptor-binding
   domain bound to the ACE2 receptor. Nature 581, 215–220 (2020).
2. Benton, D.J., Wrobel, A.G., Xu, P. et al. Receptor binding and priming of the spike
   protein of SARS-CoV-2 for membrane fusion. Nature (2020).
3. Xia, S., Liu, M., Wang, C. et al. Inhibition of SARS-CoV-2 (previously 2019-nCoV)
   infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike
   protein that harbors a high capacity to mediate membrane fusion. Cell Res 30,
   343–355 (2020).
4. https://www.ebi.ac.uk/merops/
5. https://www.uniprot.org/uniprot/P04825
6. Taq and Other Thermostable DNA Polymerases. (2008). Principles and Technical Aspects
   of PCR Amplification, 103–118. doi:10.1007/978-1-4020-6241-4\_7
7. Lissandron, V., Terrin, A., Collini, M., D’alfonso, L., Chirico, G., Pantano, S., &
   Zaccolo, M. (2005, October 21). Improvement of a FRET-based Indicator for cAMP by
   Linker Design and Stabilization of Donor–Acceptor Interaction.
8. P. Trigo-Mourino, T. Thestrup, O. Griesbeck, C. Griesinger, S. Becker, Dynamic
   tuning of FRET in a green fluorescent protein biosensor. Sci. Adv. 5, eaaw4988
   (2019).
9. Han, F., Luo, Y., Ge, N. and Xu, J. (2008), Construction of fluorescence resonance
   energy transfer vectors and their application in study of structure and function of
   signal transducers and activators of transcription 1. Acta Biochimica et Biophysica
   Sinica, 40: 934-942. doi:10.1111/j.1745-7270.2008.00480.x

A Project conducted by the 2020 NYMU iGem Team

![](https://static.igem.org/mediawiki/2020/2/2f/T--NYMU-Taipei--icon_envelope.png)
nymuigem2020@gmail.com

![](https://static.igem.org/mediawiki/2020/5/55/T--NYMU-Taipei--icon_instagram.png)
@wetlabtenpeaple

![](https://static.igem.org/mediawiki/2020/6/69/T--NYMU-Taipei--icon_facebook.png)
NYMU iGEM Team @igemnymutaipei

Copyright 2020 NYMU iGem Team. All Rights Reserved

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![](https://static.igem.org/mediawiki/2020/6/67/T--NYMU-Taipei--logo_iGem_Taipei_white.png
                        )

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[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:NYMU-Taipei/Experiments

* [Home](https://2020.igem.org/Team%3ANYMU-Taipei)
* Team

  [Members](https://2020.igem.org/Team%3ANYMU-Taipei/Team)
  [Attributions](https://2020.igem.org/Team%3ANYMU-Taipei/Attributions)
* Project

  [Description](https://2020.igem.org/Team%3ANYMU-Taipei/Description)
  [Design](https://2020.igem.org/Team%3ANYMU-Taipei/Design)
  [Results](https://2020.igem.org/Team%3ANYMU-Taipei/Results)
  [Model](https://2020.igem.org/Team%3ANYMU-Taipei/Model)
  [Software](https://2020.igem.org/Team%3ANYMU-Taipei/Software)
  [Hardware](https://2020.igem.org/Team%3ANYMU-Taipei/Hardware)
  [Implementation](https://2020.igem.org/Team%3ANYMU-Taipei/Implementation)
  [Contribution](https://2020.igem.org/Team%3ANYMU-Taipei/Contribution)
* Parts

  [Parts](https://2020.igem.org/Team%3ANYMU-Taipei/Parts)
  [Engineering](https://2020.igem.org/Team%3ANYMU-Taipei/Engineering)
* [Safety](https://2020.igem.org/Team%3ANYMU-Taipei/Safety)
* HP

  [Human Practices](https://2020.igem.org/Team%3ANYMU-Taipei/Human_Practices)
  [Collaborations](https://2020.igem.org/Team%3ANYMU-Taipei/Collaborations)
  [Education](https://2020.igem.org/Team%3ANYMU-Taipei/Education)
  [Partnership](https://2020.igem.org/Team%3ANYMU-Taipei/Partnership)
* Interlab

  [Notebook](https://2020.igem.org/Team%3ANYMU-Taipei/Notebook)
  [Experiments](https://2020.igem.org/Team%3ANYMU-Taipei/Experiments)
* Awards

  [Awards](https://2020.igem.org/Team%3ANYMU-Taipei/Awards)
  [Criteria](https://2020.igem.org/Team%3ANYMU-Taipei/Criteria)

## Experiments

![First slide](https://static.igem.org/mediawiki/2020/c/c6/T--NYMU-Taipei--experiments_display_1.jpg)

![Second slide](https://static.igem.org/mediawiki/2020/0/0d/T--NYMU-Taipei--experiments_display_2.jpg)

[Previous](#team-photos)
[Next](#team-photos)

## Plasmid Quick Extraction

1. Harvesting:
   Transfer 1.5 ml of cultured bacterial cells (1-2 x 109 E. coli grown in LB medium)
   to a 1.5 ml microcentrifuge tube. Centrifuge at 14-16,000 x g for 1 minute at room
   temperature to form a cell pellet, and then discard the supernatant completely. Use
   a narrow pipette tip to ensure the supernatant is completely removed. Repeat the
   harvesting step as required for samples between 1.5-7.0 ml using the same 1.5 ml
   microcentrifuge tube.
   NOTE: Using 2 OD600 ~ 6 OD600 units of bacterial culture is recommended. Do not use
   overgrown bacterial cultures (≤16 hours incubated in a culture tube at 37ºC with
   150-180 rpm shaking). Use fresh bacterial cultures only. Solid medium and liquid
   medium (i.e. LB medium) should contain an antibiotic such as ampicillin.
2. Resuspension:
   Add 200 µl of PD1 Buffer (make sure RNase A was added) to the 1.5 ml microcentrifuge
   tube containing the cell pellet. Resuspend the cell pellet completely by vortex or
   pipette. Continue to vortex or pipette until all traces of the cell pellet have been
   dissolved.
3. Cell Lysis:
   Add 200 µl of PD2 Buffer to the resuspended sample then mix gently by inverting the
   tube 10 times. Close PD2 Buffer bottle immediately after use to avoid CO2
   acidification. Do not vortex to avoid shearing the genomic DNA. Let stand at room
   temperature for at least 2 minutes to ensure the lysate is homogeneous. Do not
   exceed 5 minutes.
4. Neutralization:
   Add 300 µl of PD3 Buffer then mix immediately by inverting the tube 10 times. Do not
   vortex to avoid shearing the genomic DNA. Centrifuge at 14-16,000 x g for 3 minutes
   at room temperature. If using >5 ml of bacterial cells, centrifuge at 16-20,000 x g
   for 5-8 minutes. During centrifugation, place a PDH Column in a 2 ml Collection
   Tube.
5. DNA Binding:
   Transfer all of the supernatant to the PDH Column. Use a narrow pipette tip to
   ensure the supernatant is completely transferred without disrupting the white
   precipitate. Centrifuge at 14-16,000 x g for 30 seconds at room temperature then
   discard the flow-through. Place the PDH Column back in the 2 ml Collection Tube.
6. Wash:
   * For Improved Downstream Sequencing Reactions
     Add 400 µl of W1 Buffer into the PDH Column. Centrifuge at 14-16,000 x g for
     30 seconds. Discard the flow-through then place the PDH Column back in the 2
     ml Collection Tube. Proceed with Wash Buffer addition.
     NOTE: W1 Buffer is essential for efficient sequencing reactions by removing
     nuclease contamination and should be added prior to Wash Buffer addition. If
     you are not performing sequencing reactions, W1 Buffer is not required.
     Proceed directly to Wash Buffer addition.
   * For Standard Plasmid DNA Purification
     Add 600 µl of Wash Buffer (make sure absolute ethanol was added) into the
     PDH Column. Centrifuge at 14-16,000 x g for 30 seconds at room temperature.
     Discard the flow-through then place the PDH Column back in the 2 ml
     Collection Tube. Centrifuge at 14-16,000 x g for 3 minutes at room
     temperature to dry the column matrix. Transfer the dried PDH Column to a new
     1.5 ml microcentrifuge tube.
     NOTE: Perform Wash Buffer steps twice for salt sensitive downstream
     applications.
7. Elution:
   Add 50 µl of Elution Buffer1
   , TE2 or water3 into the CENTER of the column matrix. Let stand for at least 2
   minutes to allow Elution Buffer, TE or water to be completely absorbed. Centrifuge
   at 14-16,000 x g for 2 minutes at room temperature to elute the purified DNA.

## Gel extraction

1. Gel Dissociation:
   Excise the agarose gel slice containing relevant DNA fragments and remove any extra
   agarose to minimize the size of the gel slice. Transfer up to 300 mg of the gel slice to
   a 1.5 ml microcentrifuge tube. Add 500 µl of Gel/PCR Buffer and mix by vortex. Incubate
   at 60ºC for 15 minutes to ensure the gel slice has been completely dissolved. During
   incubation, invert the tube every 3 minutes. If the color of the mixture has turned to
   purple, add 10 µl of 3M Sodium Acetate (pH5.0) and mix thoroughly. Cool the dissolved
   sample to room temperature.
2. DNA Binding:
   DNA Binding
   Place a DFH Column in a 2 ml Collection Tube. Transfer 800 µl of the sample mixture to
   the DFH Column. Centrifuge at 14-16,000 x g for 30 seconds. Discard the flow-through and
   place the DFH Column back in the 2ml Collection Tube. If the sample mixture is more than
   800 µl, repeat the DNA Binding step.
3. Wash:
   Add 400 µl of W1 Buffer into the DFH Column. Centrifuge at 14-16,000 x g for 30 seconds
   then discard the flow-through. Place the DFH Column back in the 2 ml Collection Tube.
   Add 600 µl of Wash Buffer (make sure ethanol was added) into the DFH Column. Let stand
   for 1 minute at room temperature. Centrifuge at 14-16,000 x g for 30 seconds then
   discard the flow-through. Place the DFH Column back in the 2 ml Collection Tube.
   Centrifuge at 14-16,000 x g for 3 minutes to dry the column matrix.
4. DNA Elution:
   Transfer the dried DFH Column to a new 1.5 ml microcentrifuge tube. Add 25 µl of (70ºC)
   pre-heated Elution Buffer into the center of the column matrix. Let stand for at least 2
   minutes to ensure the Elution Buffer is completely absorbed. Centrifuge for 2 minutes at
   14-16,000 x g to elute the purified DNA.

## Gibson assembly

1. Set up the following reaction on ice:

   |  | 2 - 3 Fragments | 4 -6 Fragments | Positive Control |
   | --- | --- | --- | --- |
   | Total Amount of Fragments | 0.02-0.5 pmols(Xul) | 0.02-0.5 pmols(Xul) | 10 ul |
   | Master mix(2X) | 10 ul | 10 ul | 10 ul |
   | ddH2O | 10-X ul | 10-X ul | 0 ul |
   | Total Volume | 20ul | 20ul | 20ul |
2. Incubation:
   * For 2-3 fragments:
     Incubate samples in a thermocycler at 50°C for 15 minutes
   * For 4-6 Fragments:
     Incubate samples in a thermocycler at 50°C for 60 minutes
3. Post incubation:
   store samples on ice or at –20°C.

## Linear ligation

1. Set up the following reaction in a microcentrifuge tube on ice.

   | COMPONENT | 20 μl REACTION |
   | --- | --- |
   | T4 DNA Ligase Buffer | (10X)\* 2 μl |
   | Vector DNA (4 kb) | 50 ng (0.020 pmol) |
   | Insert DNA (1 kb) | 37.5 ng (0.060 pmol) |
   | Nuclease-free water | to 20 μl |
   | T4 DNA Ligase | 1 μl\* |

   * the table shows a ligation using a molar ratio of 1:3 vector to insert for the
     indicated DNA sizes.
   * The T4 DNA Ligase Buffer should be thawed and resuspended at room temperature.
2. Gently mix the reaction by pipetting up and down and microfuge briefly.
3. Stick the ends of fragments
   * For cohesive (sticky) ends:
     incubate at 16°C overnight or room temperature for 10 minutes.
   * For blunt ends or single base overhangs:
     incubate at 16°C overnight or room temperature for 2 hours
4. Heat inactivate at 65°C for 10 minutes.
5. Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells.

## 6xHis-tag Protein purification

**A. Growth of Expression Cultures:**

1. Inoculate 10 ml of 2XYT medium containing the appropriate antibiotics with a fresh
   bacterial colony harboring the expression plasmid.
2. Grow at 37°C overnight.
3. Dilute the non-induced overnight culture 1:60 (e.g., inoculate 30 ml 2XYT medium with
   500 µl overnight culture) with fresh 2XYT medium containing the appropriate antibiotics.
4. Grow at 37°C with vigorous shaking until the OD600 reaches 0.6.
5. Add IPTG to a final concentration of 1 mM and grow the culture at 37°C with vigorous
   shaking for 4 hours.IPTG(Isopropyl β-d-1-thiogalactopyranoside): MW 238.30
6. Harvest the cells by centrifugation at 6000 rpm for 15 min.

**B. Preparation (Denaturing):**

1. Buffer A:
   * 10ml: 5.73 g GuHCl;
   * 2 ml 5X Phosphate Buffer Stock Solution 1 L;
   * 573 g guanidine hydrochloride;
   * 13.80 g NaH2PO4·H2O;
   * 1.210 g Tris base6 M GuHCl;
   * 0.1 M NaH2PO4;
   * 0.01 M Tris·Cl;
   * pH 8.0
2. Buffer B - 7M urea:
   * 10ml: 3.9420 g urea;
   * 2 ml 5X Phosphate Buffer Stock Solution 1 L;
   * 394.20 g urea;
   * 13.80 g NaH2PO4·H2O;
   * 12.10 g Tris base 7 M urea;
   * 0.1 M NaH2PO4;
   * 0.01 M Tris·Cl;
   * pH 8.0
3. Buffer C:
   * 10ml: 4.8050 g urea;
   * 2 ml 5X Phosphate Buffer Stock Solution 1 L;
   * 480.50 g urea;
   * 13.80 g NaH2PO4·H2O;
   * 12.10 ag Tris base 8 M urea;
   * 0.1 M NaH2PO4;
   * 0.01 M Tris·Cl;
   * pH 6.3
4. Buffer D:
   * 10ml: 4.8050 g urea;
   * 2 ml 5X Phosphate Buffer Stock Solution 1 L;
   * 480.50 g urea;
   * 13.80 g NaH2PO4·H2O;
   * 12.10 g Tris base 8 M urea;
   * 0.1 M NaH2PO4;
   * 0.01 M Tris·HCl;
   * pH 5.9
5. Buffer E:
   * 10ml: 4.8050 g urea;
   * 2 ml 5X Phosphate Buffer Stock Solution 1 L;
   * 480.50 g urea;
   * 13.80 g NaH2PO4·H2O;
   * 12.10 g Tris base 8 M urea;
   * 0.1 M NaH2PO4;
   * 0.01 M Tris·Cl;
   * pH 4.5
6. Cell pellets frozen for at least 30 minutes at –20°C can be lysed by resuspending in
   lysis buffer and adding Benzonase® Nuclease (3 Units/ml culture volume).
7. Fresh pellets require sonication or homogenization in addition to the addition of 3
   Units/ml culture volume Benzonase® Nuclease and 1 mg/ml culture volume lysozyme

**C. Purification (Denaturing):**

1. Thaw cells for 15 min and resuspend in 700 µl Buffer B – 7 M urea and add 3 Units/ml
   culture volume Benzonase® Nuclease (i.e., for cell pellets from 5 ml cultures, add 15
   Units Benzonase® Nuclease). Resuspending pellet in 700 µl buffer will allow recovery of
   a volume of cleared lysate of approx. 600 µl.If the cells or the protein do not
   solubilize in Buffer B, then Buffer A must be used.For lower expression levels (1–5
   mg/liter), 50 ml of cell culture should be used, to give a 50x concentrated cell lysate
2. Incubate cells with agitation for 15 min at room temperature.
3. Centrifuge lysate at 10400 rpm for 15–30 min at room temperature to pellet the
   cellular debris. Collect supernatant.Save 20 µl of the cleared lysate for SDS-PAGE
   analysis.
4. Equilibrate a Ni-NTA spin column with 600 µl Buffer B – 7 M urea. Centrifuge for 2
   min at 2900 rpm. The spin columns should be centrifuged with an open lid
5. Load up to 600 µl of the cleared lysate supernatant containing the 6xHis-tagged
   protein onto a pre-equilibrated Ni-NTA spin column. Centrifuge 5 min at 1600 rpm,
   and collect the flow-through. not to exceed 1600 rpm when centrifuging. Save the
   flow-through for analysis by SDS-PAGE to check binding.
6. Wash the Ni-NTA spin column with 600 µl Buffer C. Centrifuge for 2 min at 2900
   rpm.Most proteins will remain soluble in Buffer C. If this is not the case, Buffer C
   and Buffer E should be made with 6 M guanidine hydrochloride instead of 8 M urea.For
   very low expression levels or highly concentrated lysates, 3 wash steps may be
   required to achieve high purity. Save the flow-through (wash fractions) for analysis
   by SDS-PAGE to check the stringency of the wash conditions.
7. Elute the protein twice with 200 µl Buffer E. Centrifuge for 2 min at 2900 rpm, and
   collect the eluate.Most of the 6xHis-tagged protein (>80%) should elute in the first
   200 µl, especially when proteins smaller than 30 kDa are purified. The remainder
   will elute in the second 200 µl.If higher protein concentrations are desired, do not
   combine the eluates or, alternatively, elute in 100–150 µl aliquots. If 6xHis-tagged
   monomers need to be separated from multimers, an elution step using Buffer D may be
   performed before elution with Buffer E.

## Transformation

1. Purpose:
   Transform plasmids into BL21 (Competent E. coli)
2. * BL21 competent cell
   * Plasmid DNA
   * 2XYT
   * Selection Plate
3. * Mix gently and carefully pipette 50 µl of BL21 cells into a eppendorf on ice.
   * Add 1–5 µl (containing 1 pg–100 ng) of plasmid DNA to the cell mixture.
     \*Carefully flick the tube 4–5 times to mix cells and DNA.
     \*Do not vortex.
   * Place the mixture on ice for 30 minutes.
     \*Do not mix.
   * Heat shock at exactly 42°C for exactly 30 seconds.
     \*Do not mix.
   * Place on ice for 5 minutes.
     \*Do not mix.
   * Pipette 300 µl of room temperature 2XYT into the mixture.
   * Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate. (skip if
     competent cell is weak)
   * Warm selection plates to 37°C
   * Mix the cells thoroughly by flicking the tube and inverting.
   * Spread 75 µl of each onto a selection plate(wait the plates dry) and incubate
     overnight at 37°C.
4. * Thawing:
   * Cells are best thawed on ice and DNA added as soon as the last bit of ice in the
     tube disappears. Cells can also be thawed by hand, but warming above 0°C will
     decrease the transformation efficiency.
     \*Do not vortex.
   * Incubation of DNA with Cells on Ice:
     For maximum transformation efficiency, cells and DNA should be incubated
     together on ice for 30 minutes. Expect a 2-fold loss in transformation
     efficiency for every 10 minutes you shorten this step.
   * Heat Shock:
     Both the temperature and the timing of the heat shock step are important and
     specific to the transformation volume and vessel. Using the transformation tube
     provided, 10 seconds at 42°C is optimal.
   * Outgrowth:
     Outgrowth at 37°C for 1 hour is best for cell recovery and for expression of
     antibiotic resistance. Expect a 2-fold loss in transformation efficiency for
     every 15 minutes you shorten this step. SOC medium gives 2-fold higher
     transformation efficiency than LB medium; and incubation with shaking or
     rotating the tube gives 2-fold higher transformation efficiency than incubation
     without shaking.
   * Plating:
     Selection plates can be used warm or cold, wet or dry without
     significantly affecting the transformation efficiency. However, warm, dry plates
     are easier to spread and allow for the most rapid colony formation.

## Binding analysis by western blot

**A. Sample preparation**

1. Remove a small volume of lysate to perform a protein quantification assay. Determine
   the protein concentration for each cell lysate.
2. Determine how much protein to load (60 ng) and add an equal volume 2X Laemmli sample
   buffer.​
3. To reduce and denature samples, boil each cell lysate in sample buffer at 100°C for
   5 min. Lysates can be aliquoted and stored at -20°C for future use.

**B. Loading and running the gel**

1. Load equal amounts of protein into the wells of the SDS-PAGE gel, along with
   molecular weight marker. Load 60 ng of purified protein.
2. Run the gel for 1–2 h at 100 V.
3. Transferring the protein from the gel to the membrane
4. Block the membrane for 1 h at room temperature or overnight at 4°C using blocking
   buffer.
5. Incubate the membrane with appropriate dilutions of primary antibody in blocking
   buffer. We recommend overnight incubation at 4°C
6. Wash the membrane in three washes of TBST, 5 min each
7. Incubate the membrane with the recommended dilution of conjugated secondary antibody
   in blocking buffer at room temperature for 1 h.
8. Wash the membrane in three washes of TBST, 5 min each.
9. For signal development, follow the kit manufacturer’s recommendations. Remove excess
   reagent and cover the membrane in transparent plastic wrap.
10. Acquire image using darkroom development techniques for chemiluminescence, or normal
    image scanning methods for colorimetric detection.

## Hydrogel making and protein immoblization

**A. Hydrogel making**

1. Add 13ml HEMA((2-hydroxyethyl methacrylate) and 5ml H2O to mixed solution and keep
   stir for 5min
2. Add 0.02g TEGDMA to mixed solution then stir for 20min
3. Add 5ml H2O and 0.06g AIBN to beaker, then stir for 5min
4. Go microwave for 30 sec
5. Distribute to five 5cm plates
6. Put Five in the dry oven (temperature: 90 degrees Celcius) for 2hours

**B. Protein immoblization**

1. Prepare 0.05% KMnO4 solution
2. Drip the solution on the surface of pHEMA for a while to oxidize -OH group (the
   changing of solution from purple to brown means the interaction completed)
3. use Fourier-transform infrared spectroscopy (FTIR)to check whether the functional
   group had change(the peak of C、O double bond shoud be higher under same background
   after oxidation).
4. Soak hydrogel into Cu2+ contained aqueous solution(0.03g CuSO4 + 10g NaOH solved in
   100ml water) mechanically stirred at room temperature for 2 hours make Cu2+ chelated
   with -COOH to make a Ni-NTA alike surface
5. Soaking hydrogel into aqueous solution which contains proteins(Xaa-Pro aminopeptide
   with N-terminal 6X His tag) and mechanically stirred at room temperature for 2 hours
   to make 6X His-tag chelated with Cu2+ which results Xaa-Pro aminopeptides properly
   covered on the surface of polyHEMA.

A Project conducted by the 2020 NYMU iGem Team

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nymuigem2020@gmail.com

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@wetlabtenpeaple

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NYMU iGEM Team @igemnymutaipei

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# Team:NYMU-Taipei/Results

* [Home](https://2020.igem.org/Team%3ANYMU-Taipei)
* Team

  [Members](https://2020.igem.org/Team%3ANYMU-Taipei/Team)
  [Attributions](https://2020.igem.org/Team%3ANYMU-Taipei/Attributions)
* Project

  [Description](https://2020.igem.org/Team%3ANYMU-Taipei/Description)
  [Design](https://2020.igem.org/Team%3ANYMU-Taipei/Design)
  [Results](https://2020.igem.org/Team%3ANYMU-Taipei/Results)
  [Model](https://2020.igem.org/Team%3ANYMU-Taipei/Model)
  [Software](https://2020.igem.org/Team%3ANYMU-Taipei/Software)
  [Hardware](https://2020.igem.org/Team%3ANYMU-Taipei/Hardware)
  [Implementation](https://2020.igem.org/Team%3ANYMU-Taipei/Implementation)
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* Parts

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* HP

  [Human Practices](https://2020.igem.org/Team%3ANYMU-Taipei/Human_Practices)
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  [Partnership](https://2020.igem.org/Team%3ANYMU-Taipei/Partnership)
* Interlab

  [Notebook](https://2020.igem.org/Team%3ANYMU-Taipei/Notebook)
  [Experiments](https://2020.igem.org/Team%3ANYMU-Taipei/Experiments)
* Awards

  [Awards](https://2020.igem.org/Team%3ANYMU-Taipei/Awards)
  [Criteria](https://2020.igem.org/Team%3ANYMU-Taipei/Criteria)

### Results

---

#### ACE2-Spike Binding Affinity

##### Plasmid construction

**ACE2**

We successfully constructed a plasmid that has ACE2 RBD coding sequence with a
bacterial expression backbone and 6x histag.

![](https://static.igem.org/mediawiki/2020/9/9c/T--NYMU-Taipei--result_ACE2_schematic.png)
![](https://static.igem.org/mediawiki/2020/e/e6/T--NYMU-Taipei--result_ACE2.png
                                    )

**plasmid overview on snapgene(3953bp)
gel electrophoresis digested by BamHI(1061) and EcoRI(1791)**

---

#### Protease Cleaving Activity

##### Plasmid construction

**pepP**

We successfully constructed a plasmid that has pepP coding sequence with a bacterial
expression backbone and 6x histag.

![](https://static.igem.org/mediawiki/2020/4/45/T--NYMU-Taipei--result_pepP_schematic.png                                    )
![](https://static.igem.org/mediawiki/2020/5/5f/T--NYMU-Taipei--result_pepP.png)

**plasmid overview on snapgene(3056bp)
gel electrophoresis digested by XbaI(70) and HindIII(1590)**

we expressed the construct through E. coli Bl21, purify with Ni-NTA column and
tested the correctness through nanodrop, SDS-page and Western blotting.

**Nanodrop result and concentration(protein
presented in the A280 peak\***

![](https://static.igem.org/mediawiki/2020/c/cc/T--NYMU-Taipei--result_fig1.jpg)
![](https://static.igem.org/mediawiki/2020/4/42/T--NYMU-Taipei--result_fig2.jpg
                                        )
![](https://static.igem.org/mediawiki/2020/f/fe/T--NYMU-Taipei--result_fig3.jpg
                                        )

**\*Note that the concentration is different from
the experiment concentration since the elution buffer is full of imidazo. We
use an exchance membrane to remove them to present interference of
experimental condition.**

**SDS-page result**

![](https://static.igem.org/mediawiki/2020/3/3c/T--NYMU-Taipei--result_SDS-page_result.png
                                        )

**Western Blot result**

![](https://static.igem.org/mediawiki/2020/d/dc/T--NYMU-Taipei--result_Western_Blot_result.png
                                        )

**spike fragments**

We constructed four fragments from SARS-CoV-2 spike protein(4.2kDa each) with a pepP
cleavage site on each of them to test the cleavage activity (1st fragment comes up
with a unfavored result).

![](https://static.igem.org/mediawiki/2020/a/ab/T--NYMU-Taipei--result_spike_fragments_schematic.png
                                    )
![](https://static.igem.org/mediawiki/2020/5/50/T--NYMU-Taipei--result_spike_fragments.png
                                    )

**plasmid overview on snapgene(1799bp)
gel electrophoresis digested by XbaI(70) and HindIII(333)**

We also expressed the construct through E. coli Bl21, purify with Ni-NTA column and
tested the correctness through nanodrop and SDS-page. Special tricine SDS page was
used here since the protein is only 4kDa.

---

#### Protease-ACE2 Cleaving Activity

##### Plasmid construction

**pepP-ACE2(P+A)**

We successfully constructed a plasmid that has pepP + ACE2 RBD coding sequence with
a bacterial expression backbone and 6x histag

![](https://static.igem.org/mediawiki/2020/6/6d/T--NYMU-Taipei--result_pepP-ACE2%28P%2BA%29_schematic.png
                                    )
![](https://static.igem.org/mediawiki/2020/8/8d/T--NYMU-Taipei--result_pepP-ACE2%28P%2BA%29.png
                                    )

**plasmid overview on snapgene(5312bp)
gel electrophoresis digested by XbaI(5270) and NheI(1329)**

**spike fragments**

We are using the same spike fragments as pepP cleaving activity.

---

#### Gel conjugation

##### polyHEMA making

**pepP-ACE2(P+A)**

Because polyHEMA is a mesh aggregation of thermal polymerization, we experieced
different heat condition to find out the best product method.

The result showed that althought microwave oven is much faster than dry oven, it is
hard for us to control the stability of temperature of our gel. Instead, if we use
dry oven in 75-85 degree Celsius, the gel will easily reach the perfact situation,
which is jelly-like status and without bubble or withering.

\*the heat mentioned above is the step after heating in microwave
oven.

**75 degree Celsius in dry oven for 2
hours**

![](https://static.igem.org/mediawiki/2020/d/d4/T--NYMU-Taipei--result_polyHEMA_making_fig1.jpg
                                        )

**Microwave for 25 seconds**

![](https://static.igem.org/mediawiki/2020/f/f0/T--NYMU-Taipei--result_polyHEMA_making_fig2.jpg
                                        )

**Microwave for 20 seconds**

![](https://static.igem.org/mediawiki/2020/7/79/T--NYMU-Taipei--result_polyHEMA_making_fig3.jpg
                                        )

**Microwave for 15 seconds**

![](https://static.igem.org/mediawiki/2020/6/68/T--NYMU-Taipei--result_polyHEMA_making_fig4.jpg)

**Microwave for 10 seconds**

![](https://static.igem.org/mediawiki/2020/1/1e/T--NYMU-Taipei--result_polyHEMA_making_fig5.jpg)

**Oxidation of polyHEMA**

We use Potassium permanganate to oxidase the hydroxyl group on polyHEMA. After
oxidation, we used FTIR to detect whether the functional group on polyHEMA had
changed from R-OH to R-COOH. The result indicated that the site of classical
carboxyl group adsorbing had a obvious increase.

![](https://static.igem.org/mediawiki/2020/1/17/T--NYMU-Taipei--result_Oxidation_of_polyHEMA.jpg
                                    )

A Project conducted by the 2020 NYMU iGem Team

![](https://static.igem.org/mediawiki/2020/2/2f/T--NYMU-Taipei--icon_envelope.png)
nymuigem2020@gmail.com

![](https://static.igem.org/mediawiki/2020/5/55/T--NYMU-Taipei--icon_instagram.png)
@wetlabtenpeaple

![](https://static.igem.org/mediawiki/2020/6/69/T--NYMU-Taipei--icon_facebook.png)
NYMU iGEM Team @igemnymutaipei

Copyright 2020 NYMU iGem Team. All Rights Reserved

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![](https://static.igem.org/mediawiki/2020/6/67/T--NYMU-Taipei--logo_iGem_Taipei_white.png
                        )

![](https://static.igem.org/mediawiki/2020/3/3b/T--NYMU-Taipei--sponsor_snapgene_white.png)