iGEM 2020 — Team Korea_HS

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# Team:Korea HS/Notebook

# [KOREA\_HS](https://2020.igem.org/Team%3AKorea_HS)

[JUDGING](https://igem.org/2020_Judging_Form?team=Korea_HS)

* [TEAM](https://2020.igem.org/Team%3AKorea_HS/Team)

  + [Team](https://2020.igem.org/Team%3AKorea_HS/Team)
  + [Attributions](https://2020.igem.org/Team%3AKorea_HS/Attributions)
  + [Collaborations](https://2020.igem.org/Team%3AKorea_HS/Collaborations)
  + [Inclusion](https://2020.igem.org/Team%3AKorea_HS/Inclusion)
* [PROJECT](https://2020.igem.org/Team%3AKorea_HS/Contribution)

  + [Contribution](https://2020.igem.org/Team%3AKorea_HS/Contribution)
  + [Description](https://2020.igem.org/Team%3AKorea_HS/Description)
  + [Design](https://2020.igem.org/Team%3AKorea_HS/Design)
  + [Engineering](https://2020.igem.org/Team%3AKorea_HS/Engineering)
  + [Experiments](https://2020.igem.org/Team%3AKorea_HS/Experiments)
  + [Notebook](https://2020.igem.org/Team%3AKorea_HS/Notebook)
  + [Partnership](https://2020.igem.org/Team%3AKorea_HS/Partnership)
  + [Results](https://2020.igem.org/Team%3AKorea_HS/Results)
  + [Implementation](https://2020.igem.org/Team%3AKorea_HS/Implementation)
* [PARTS](https://2020.igem.org/Team%3AKorea_HS/Part_Collection)

  + [Part Collection](https://2020.igem.org/Team%3AKorea_HS/Part_Collection)
  + [Parts](https://2020.igem.org/Team%3AKorea_HS/Parts)
* [SAFETY](https://2020.igem.org/Team%3AKorea_HS/Safety)

  + [Safety](https://2020.igem.org/Team%3AKorea_HS/Safety)
* [HUMAN PRACTICES](https://2020.igem.org/Team%3AKorea_HS/Human_Practices)

  + [Human Practices](https://2020.igem.org/Team%3AKorea_HS/Human_Practices)
* [AWARDS](https://2020.igem.org/Team%3AKorea_HS/Education)

  + [Education](https://2020.igem.org/Team%3AKorea_HS/Education)
  + [Model](https://2020.igem.org/Team%3AKorea_HS/Model)
  + [Medal Criteria](https://2020.igem.org/Team%3AKorea_HS/Medal_Criteria)

## KOREA\_HS

Designing a Hyperstable Antibody with
Cell-penetrating Peptide for Intracellular Targeting

* [Contribution](https://2020.igem.org/Team%3AKorea_HS/Contribution)|  |
* [Description](https://2020.igem.org/Team%3AKorea_HS/Description)|  |
* [Design](https://2020.igem.org/Team%3AKorea_HS/Design)|  |
* [Engineering](https://2020.igem.org/Team%3AKorea_HS/Engineering)|  |
* [Experiments](https://2020.igem.org/Team%3AKorea_HS/Experiments)|  |
* [Notebook](https://2020.igem.org/Team%3AKorea_HS/Notebook)|  |
* [Partnership](https://2020.igem.org/Team%3AKorea_HS/Partnership)|  |
* [Results](https://2020.igem.org/Team%3AKorea_HS/Results)|  |
* [Implementation](https://2020.igem.org/Team%3AKorea_HS/Implementation)|  |

## Notebook

|  |
| --- |
| Whole Team 12 March 2020  Team Korea\_HS created  21 March 2020  1st zoom orientation meeting  : Initial team members met for the first time. We took a look at medal criteria as a team. The professor briefly explained last year’s experiment.  4 April 2020  2nd zoom meeting  : The professor presented a detailed explanation for last year’s project. We talked about our goal for the project.  25 April 2020  3rd zoom meeting  : Last year’s Korea\_HS team members (Dyanne, Sunwoo, Bruce) joined the zoom call and shared their iGEM experience. We checked changes iGEM made due to pandemic. We divided the team into three sub-teams: human practice, collaboration, and wiki. Team members chose leaders for the team.  23 May 2020  4th zoom meeting  : Each sub-team presented their goals for this competition.  13 June 2020  1st in-person meeting  : Me each other for the first time in person. Talked about last year’s experiment and what changes we are going to make this year for an improvement.  2 August 2020  2nd in-person meeting  : Took individual portraits and team pictures. Also, our student mentor Dyanne explained about last year’s experiment and gave tips and answered questions  9 August 2020  3rd in-person meeting  : Had a zoom meeting with Professor Jungwoo Choe and talked about modeling and assay. After the zoom session, each sub-team held their own meeting for an hour.  23 August 2020  4th in-person meeting  : Had team lunch and each sub-team had their own discussion for two hours.  13 September 2020  5th zoom meeting  : Finalized individual bio and chose pictures that will be to used for wiki  20 September 2020  6th zoom meeting  : Distributed parts for presentation slides rough draft and brainstormed ideas for the general theme and color of wiki  27 September 2020  7th zoom meeting  : Set plans for wiki building and created folders for each menu and tab  4 October 2020  8th zoom meeting  : Finalized the rough draft for presentation slides, and chose members to present  11 October 2020  9th zoom meeting  : Went over uncompleted informations required for wiki building, built and designed the homepage and tabs on the website  18 October 2020  10th zoom meeting  : Set plans for presentation and poster making, finalized informations used for wiki building  25 October 2020  11th zoom meeting  : Went over the design and layout for wiki, distributed parts for the Presentation Video Experiment 5 July 2020  1st Lab  : PCR amplification of insert HRas G12V and scFv(Ras)  12 July 2020  2nd Lab  : Transformation and recombination of plasmid to E.coli  19 July 2020  3rd Lab  : Purification of histidine tagged scFv(Ras) and HRas  23 July 2020  4th Lab  : SDS-PAGE of purified histidine-tagged scFv(Ras) and HRas  26 July 2020  5th Lab  : Western Blotting  30 July 2020  Assay  : Binding assay of scFv(Ras) and HRas Collaboration 31 May 2020  1st collaboration team meeting  : Collaboration team members met for the first time  28 June 2020  1st meeting with UNILausanne  : Each team explained their project and wet lab plans  5 July 2020  1st meeting with DTU Denmark  : Each team explained their project and plans for public engagement  12 July 2020  1st meeting with Queen’s University  : Each team introduced their project and gave lab suggestions  18 July 2020  1st meeting with Imperial College London  : Participate in their mathematical modelling outreach, exchange about project progress  19 July 2020  1st meeting with NJU China  : Exchanged team projects and inspirations. Also, received great tips on public engagement as they told us they are writing a story for children about tumor  26 July 2020  1st iGEM mentorship meeting  : Share about our project and receive feedback on current progress with human practices work  1 August 2020  2nd iGEM mentorship meeting  : Questions prepared prior to the meeting were answered, and Mentor Saniya gave us tips on presentation with examples  2 August 2020  2nd meeting with DTU Denmark  : Exchanged tips on wiki and poster building  8 August 2020  Korea Meetup  : Team KSA\_KOREA, Korea\_HS, and Korea-SIS held an offline meeting. Introduced each other, explained projects, and exchanged questions.  9 August 2020  2nd meeting with Queen’s University  : Received suggestions for the cell penetration assay and binding assay  15 August 2020  2nd meeting with Imperial College London  : Learned the purpose for mathematical modeling and tips on homology modeling. Taught us that modeling should go beyond our experiment.  22 August 2020  2nd meeting with UNILausanne  : Discussed about modeling and wiki building  25 September 2020  2nd meeting with NJU China  : Discussed about WIKI guidelines and finalizing our projects.  10 October 2020  3rd Meeting with DTU Denmark  :Discussed about adding images to WIKI website and the learning experiences of IGEM2020 Human Practices 15 July 2020  Interview with CELLID Discussed lab procedures and how to solve problems we faced.  21 July 2020  Interview with GENESCO  :Discussed advantages and disadvantages of our treatment  5 August 2020  Interview with ORUMRX Discussed real life applications and affected population from our research  10 August 2020  First survey sent out to raise awareness for our project  13 September 2020  Jina entered the KSCY  : The KSCY competition became a platform to share our project with other ambitious students and college professors. Due to the coronavirus, the seminar took place online.  22 September 2020  Pamphlet created by Yoonyee and Melissa with information on our project and antibody therapeutics.  24 September 2020  Schoolwide presentation about our project and what it is, and its potential implications and importance on the students.  29 September 2020  Pamphlet distribution by Yeonjo, informing her peers about the project and its goals, and inquiring their existing knowledge in antibody therapeutics.  03 October 2020  Pamphlet distributed around the school by Melissa to 9th graders, to inform them of our project.  16 October 2020  Second survey sent out, inquiring them about their previous knowledge in antibody related therapeutics, and their perceived dangers of these therapeutics. |

2020 By iGEM team : Korea\_HS / e-mail : igem.koreahs@gmail.com
Copyrights(c) 2020 KORHS(KOREA\_HS) ALL RIGHTS RESERVED.

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

# Team:Korea HS/Engineering

# [KOREA\_HS](https://2020.igem.org/Team%3AKorea_HS)

[JUDGING](https://igem.org/2020_Judging_Form?team=Korea_HS)

* [TEAM](https://2020.igem.org/Team%3AKorea_HS/Team)

  + [Team](https://2020.igem.org/Team%3AKorea_HS/Team)
  + [Attributions](https://2020.igem.org/Team%3AKorea_HS/Attributions)
  + [Collaborations](https://2020.igem.org/Team%3AKorea_HS/Collaborations)
  + [Inclusion](https://2020.igem.org/Team%3AKorea_HS/Inclusion)
* [PROJECT](https://2020.igem.org/Team%3AKorea_HS/Contribution)

  + [Contribution](https://2020.igem.org/Team%3AKorea_HS/Contribution)
  + [Description](https://2020.igem.org/Team%3AKorea_HS/Description)
  + [Design](https://2020.igem.org/Team%3AKorea_HS/Design)
  + [Engineering](https://2020.igem.org/Team%3AKorea_HS/Engineering)
  + [Experiments](https://2020.igem.org/Team%3AKorea_HS/Experiments)
  + [Notebook](https://2020.igem.org/Team%3AKorea_HS/Notebook)
  + [Partnership](https://2020.igem.org/Team%3AKorea_HS/Partnership)
  + [Results](https://2020.igem.org/Team%3AKorea_HS/Results)
  + [Implementation](https://2020.igem.org/Team%3AKorea_HS/Implementation)
* [PARTS](https://2020.igem.org/Team%3AKorea_HS/Part_Collection)

  + [Part Collection](https://2020.igem.org/Team%3AKorea_HS/Part_Collection)
  + [Parts](https://2020.igem.org/Team%3AKorea_HS/Parts)
* [SAFETY](https://2020.igem.org/Team%3AKorea_HS/Safety)

  + [Safety](https://2020.igem.org/Team%3AKorea_HS/Safety)
* [HUMAN PRACTICES](https://2020.igem.org/Team%3AKorea_HS/Human_Practices)

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* [AWARDS](https://2020.igem.org/Team%3AKorea_HS/Education)

  + [Education](https://2020.igem.org/Team%3AKorea_HS/Education)
  + [Model](https://2020.igem.org/Team%3AKorea_HS/Model)
  + [Medal Criteria](https://2020.igem.org/Team%3AKorea_HS/Medal_Criteria)

## KOREA\_HS

Designing a Hyperstable Antibody with
Cell-penetrating Peptide for Intracellular Targeting

* [Contribution](https://2020.igem.org/Team%3AKorea_HS/Contribution)|  |
* [Description](https://2020.igem.org/Team%3AKorea_HS/Description)|  |
* [Design](https://2020.igem.org/Team%3AKorea_HS/Design)|  |
* [Engineering](https://2020.igem.org/Team%3AKorea_HS/Engineering)|  |
* [Experiments](https://2020.igem.org/Team%3AKorea_HS/Experiments)|  |
* [Notebook](https://2020.igem.org/Team%3AKorea_HS/Notebook)|  |
* [Partnership](https://2020.igem.org/Team%3AKorea_HS/Partnership)|  |
* [Results](https://2020.igem.org/Team%3AKorea_HS/Results)|  |
* [Implementation](https://2020.igem.org/Team%3AKorea_HS/Implementation)|  |

## Engineering

|  |
| --- |
| Dialysis and TEV Cut Procedure The Dialysis and TEV Cut was executed after Wester blotting, which confirmed that the solution contained HRas G12V. The purpose of dialysis and TEV cut was to get a pure/independent HRas G12V without extraneous parts. For the experiment, from the start, HRas G12V was tagged with Histidine to act as an indicator of HRas G12V during Ni-NTA Affinity Chromatography since Histidine binds with Nickel. Therefore TEV protease cut HRas G12Vand the Histidine tagged, leaving pure HRas G12V. At the same time, dialysis was performed to get rid of imidazole. Since imidazole was used to wash separated HRas G12V from the agarose gel, imidazole was in the HRas G12V solution. Imidazole limited TEV protease function, therefore, must be removed. After, another Ni-NTA affinity chromatography was done to separate the TEV protease and the detached histidine tag from the HRas G12V solution. It was possible because TEV proteases have Histidine.  To verify that the experiment was a success (that TEV protease cut the Histidine tag of most of the Histidine tagged HRas G12V) the protein was transferred onto columns for SDS-Page. Results The expected result was a distinct line between 20-25 kDa just a little bit below the line shown on the uncut variable since HRas G12V is 21.4kDa. Unexpectedly the SDS-Page results came out as a smudge with several different small lines, instead of a line making it impossible to verify whether or not the solution was composed of pure HRas G12V.  ![](https://static.igem.org/mediawiki/2020/e/e2/T--Korea_HS--eg1.png) Possibilities There is no definite cause for the unclear result, however, there are several possibilities.    Contaminated Solution :The solution inserted in the SDS-Page columns might have been contaminated with other proteins/contaminants. This explains the numerous small strands shown on the ‘Cut’ column which is supposed to show one distinct line between 20-25kDa that indicates HRas G12V. Such contaminants can be traced back to the protein samples being kept under poor conditions. Band Pattern :The smudged lines shown on the graph can be the natural line.  Solution Since there are so many possible reasons why SDS-Page results came out in a smudge, we decided to verify the transferred membrane by requesting sequencing. It is nearly impossible to find out the exact reason as to why there was a smudge unless a very apparent clue comes out during the process. It was also very likely to fail again because we would repeat the same process without knowing the exact reason. Therefore, we concluded that it would be more time and cost-effective to request our transferred membrane sequencing. Results As an alternative solution to verify that the protein solution contains pure HRas G12V, we sent the protein sample to a company that sequenced the given sample through HPLC chromatography. Thankfully the sequenced protein was identical to the sequence of HRas G12V, which verified that the protein was purified HRas G12V that we were looking for. What We Learned From this process, we learned that finding an alternate method can sometimes be more effective than holding onto just one type of method. If we only stuck to Western blot and redid the process without sequencing until we got precise results, we would have discarded the wanted protein. Also, we could have spent days figuring out the exact reason why the results came out unclear. Without knowing the failure's specific cause, it is always good to seek other verification methods. |

2020 By iGEM team : Korea\_HS / e-mail : igem.koreahs@gmail.com
Copyrights(c) 2020 KORHS(KOREA\_HS) ALL RIGHTS RESERVED.

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

# Team:Korea HS/Experiments

# [KOREA\_HS](https://2020.igem.org/Team%3AKorea_HS)

[JUDGING](https://igem.org/2020_Judging_Form?team=Korea_HS)

* [TEAM](https://2020.igem.org/Team%3AKorea_HS/Team)

  + [Team](https://2020.igem.org/Team%3AKorea_HS/Team)
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  + [Inclusion](https://2020.igem.org/Team%3AKorea_HS/Inclusion)
* [PROJECT](https://2020.igem.org/Team%3AKorea_HS/Contribution)

  + [Contribution](https://2020.igem.org/Team%3AKorea_HS/Contribution)
  + [Description](https://2020.igem.org/Team%3AKorea_HS/Description)
  + [Design](https://2020.igem.org/Team%3AKorea_HS/Design)
  + [Engineering](https://2020.igem.org/Team%3AKorea_HS/Engineering)
  + [Experiments](https://2020.igem.org/Team%3AKorea_HS/Experiments)
  + [Notebook](https://2020.igem.org/Team%3AKorea_HS/Notebook)
  + [Partnership](https://2020.igem.org/Team%3AKorea_HS/Partnership)
  + [Results](https://2020.igem.org/Team%3AKorea_HS/Results)
  + [Implementation](https://2020.igem.org/Team%3AKorea_HS/Implementation)
* [PARTS](https://2020.igem.org/Team%3AKorea_HS/Part_Collection)

  + [Part Collection](https://2020.igem.org/Team%3AKorea_HS/Part_Collection)
  + [Parts](https://2020.igem.org/Team%3AKorea_HS/Parts)
* [SAFETY](https://2020.igem.org/Team%3AKorea_HS/Safety)

  + [Safety](https://2020.igem.org/Team%3AKorea_HS/Safety)
* [HUMAN PRACTICES](https://2020.igem.org/Team%3AKorea_HS/Human_Practices)

  + [Human Practices](https://2020.igem.org/Team%3AKorea_HS/Human_Practices)
* [AWARDS](https://2020.igem.org/Team%3AKorea_HS/Education)

  + [Education](https://2020.igem.org/Team%3AKorea_HS/Education)
  + [Model](https://2020.igem.org/Team%3AKorea_HS/Model)
  + [Medal Criteria](https://2020.igem.org/Team%3AKorea_HS/Medal_Criteria)

## KOREA\_HS

Designing a Hyperstable Antibody with
Cell-penetrating Peptide for Intracellular Targeting

* [Contribution](https://2020.igem.org/Team%3AKorea_HS/Contribution)|  |
* [Description](https://2020.igem.org/Team%3AKorea_HS/Description)|  |
* [Design](https://2020.igem.org/Team%3AKorea_HS/Design)|  |
* [Engineering](https://2020.igem.org/Team%3AKorea_HS/Engineering)|  |
* [Experiments](https://2020.igem.org/Team%3AKorea_HS/Experiments)|  |
* [Notebook](https://2020.igem.org/Team%3AKorea_HS/Notebook)|  |
* [Partnership](https://2020.igem.org/Team%3AKorea_HS/Partnership)|  |
* [Results](https://2020.igem.org/Team%3AKorea_HS/Results)|  |
* [Implementation](https://2020.igem.org/Team%3AKorea_HS/Implementation)|  |

## Experiments

|  |
| --- |
| 1st Lab5 July 2020PCR amplification of insert (HRas G12V)  1. PCR amplification of scFv(Ras) insert (805 bp)    1. Set up PCR reactions and prepare to run PCR program    2. PCR Mix        : Apply (50 μL total)       * 42 μL of sterile ddH2O       * 5 μL of reaction buffer (10x)       * Try Pfu first and if it does not work, try Vent       * 0.5 μL of dNTP mix at 2.5 mM       * 0.5 μL of forward primer at 100 μM       * 0.5 μL of reverse primer at 100 μM       * 1 μL of genomic or plasmid DNA       * 0.5 μL of DNA polymerase    3. PCR Program       1. Initial Denaturation at 94ºC – 5 min       2. Denaturation at 94ºC – 1 min       3. Annealing at 55ºC – 30 sec       4. Extension at 72ºC – 1 min./kb target sequence       5. Go to steps ii through iv again (25 times)       6. Final Extension at 72ºC – 5 min       7. Storage at 4ºC – ∞ 2. Check the PCR product with DNA gel electrophoresis.    1. Dissolve 0.36 g agarose in 30 mL TAE buffer (1x)    2. Microwave the mixture to a boil, and be careful not to boil over    3. Cool down to a point below 60℃ and add 0.6 ㎕ of EtBr    4. EtBr is very dangerous so make sure to wear gloves when handling the chemical    5. Stir well and pour it to a gel cast    6. Insert the comb in the gel cast and let the gel solidify for 30 minutes    7. Remove the comb and apply samples from PCR    8. Run gel electrophoresis for 15 min at 120V    9. Check if HRas G12V insert (610 base pairs) is present   ![](https://static.igem.org/mediawiki/2020/d/d4/T--Korea_HS--lab1.png)  2nd Lab12 July 2020Transformation and recombination of the plasmid (LIC vector - scFv(Ras)) to E. coli competent cell (BL21(DE)3) B strains are deficient in Lon protease (cytoplasm) and OmpT protease (outer membrane). Therefore, B strains are generally preferred for recombinant protein expression. The DE3 strains carry the gene for T7 RNA polymerase under the control of the lacUV5 promoter. IPTG is required to maximally induce expression of the T7 RNA polymerase in order to express recombinant genes cloned downstream of a T7 promoter. BL21(DE3) is suitable for expression from a T7 or T7-lac promoter or promoters recognized by the E.coli RNA polymerase.   1. Create a mixture using 20 ㎕ of competent cell and 1.5 ㎕ of DNA (LIC vector - scFv(Ras)) 2. Put the mixture in ice for 20 minutes 3. Apply heat shock for 1 minute at 42℃ 4. Put the mixture back in ice for 20 minutes 5. Add 200 ㎕ of LB, shaking incubation at 37℃ for one hour at 200 rpm 6. Make sure to label and preheat LB plate 7. Prepare a tabletop centrifuge and centrifuge for 3 minutes at 3,000 rpm 8. Discard 150 ㎕ of the supernatant by pouring or using a pipette 9. Resuspend the cell pellets by gentle tapping 10. Start plating by streaking and put the plates in an incubator at 37℃ overnight   ![](https://static.igem.org/mediawiki/2020/c/cb/T--Korea_HS--lab2.png)  3rd Lab19 July 2020Purification of histidine-tagged scFv(Ras) using Ni2+-NTA resin affinity chromatography  1. Prepare soluble fraction of cell lysate –SOL sample collect (10 ㎕) 2. Equilibration: 20 mM Tris-HCl pH 7.5 + 250 mM NaCl buffer 5CV (column volume) 3. Loading through: soluble fraction of cell lysate –LT sample collect (10 ㎕) 4. Wash: buffer + 20 mM imidazole 5CV –WA sample collect (20 ㎕) 5. Elution 1: buffer + 100 mM imidazole 5mL - 100mM sample collect (20 ㎕) 6. Elution 2: buffer + 400 mM imidazole 5mL - 400mM sample collect (20 ㎕)   ![](https://static.igem.org/mediawiki/2020/7/7a/T--Korea_HS--lab3.png)  4th Lab23 July 2020SDS-PAGE of purified histidine-tagged (scFv(Ras)) SDS-PAGE is a method of separating proteins based on their molecular mass. SDS(sodium dodecyl sulfate) is a detergent that binds proteins and covers them with a negative charge. After exposure to SDS different proteins will have a very similar charge to mass ratios because SDS coats the protein in a negative charge overwhelming whatever intrinsic charge the protein originally had. The denatured protein mixture is added to a polyacrylamide gel where an electric field is applied and the proteins move towards the positive electrode based on their size. The upper gel(stacking gel) first allows proteins to align the top of the resolving gel, then resolving gel separate proteins on their molecular weight.   1. Preparing SDS-PAGE loading samples    1. Add 3’DW 10 ㎕ + 6x dye 4 ㎕ for SOL, LT samples    2. Add 6x dye 4 ㎕ for WA, 100 mM, 400 mM samples.    3. Boil the sample for 5 minutes.    4. Check the scFv(Ras) band through SDS-PAGE 2. Preparing SDS-PAGE gel    1. Assemble the gel casting apparatus, making sure that the sandwich of glass plates makes a good seal.    2. Prepare 5 mL of separating gel solution according to the 12% acrylamide concentration recipe. Mix thoroughly.    3. Fill the apparatus with separating gel solution, leaving about 2 cm of space for the stacking gel.    4. Fill the apparatus with around 1 mL of 2-propanol to isolate the polymerization reaction from oxygen and flatten the separating gel.    5. Give around 20 minutes for the separating gel to solidify.    6. After the separating gel solidifies, pour off the 2-propanol and rinse with distilled water.    7. Remove any water droplets from the inside of the casting apparatus with Whatman filter paper.    8. Prepare 2 mL of 5% stacking gel solution. Mix thoroughly.    9. Fill the stacking gel solution to the top. Place the 10-well comb into the apparatus without introducing air bubbles.    10. Allow the stacking gel to polymerize completely for around 20 minutes before removing the comb.    11. Prepare the samples i) Mix 15 ㎕ protein samples with 3 ㎕ of SDS Sample Buffer (6x)         ii) Prepare the following samples (crude, loading through, wash, elution)         iii) Heat the samples for 5 minutes in boiling water    12. Clip the sandwich to the electrophoresis apparatus. Carefully remove the comb from the gel and fill the inside of the apparatus with an SDS running buffer (1x).    13. Carefully load 5 ㎕ of the protein MW marker and 10 ㎕ of samples (soluble, insoluble, loading thru, wash, 100 mM, 400 mM elution fractions) into the bottom of the wells using P20 pipette.    14. Fill the outside of the electrophoresis apparatus with SDS running buffer (1x) and connect the apparatus to the power supply.    15. Run the gel at 150V for around 60 minutes (one hour) until the dye reaches the bottom of the separating gel.    16. Turn off the power supply, and remove the gel sandwich.    17. Carefully open the sandwich by using one of the spacers to pry the plates apart.    18. Put the gel in about 20 mL of Coomassie staining solution. Shake gently for at least 30 minutes.    19. Pour the staining solution back to the bottle and destain the gel using around 100 mL of the destaining solution for at least 30 minutes.    20. Pour off the destaining solution and put the gel in water.    21. Take a picture of the gel  5th Lab30 July 2020Western blotting  1. Prepare 1 L of 1X transfer buffer mix (680 ml dH2O + 170 ml 5X transfer buffer + 150 ml MeOH) 2. Assemble the transfer sandwich (sponge – paper – gel – membrane – paper – sponge). Make sure the PVDF membrane should be placed on MeOH for ~1 min to activate and no air bubbles are trapped in the sandwich. The 3. membrane should be on the cathode and the gel on the anode. 4. Place the cassette in the transfer tank and place an ice block in the tank. 5. Transfer the gel for 60 min at 100 V. 6. Transferred membranes should be placed on MeOH and dry to remove residual alcohol. 7. Incubate membrane in TBS-T buffer at 4 oC for the next experiment. 8. Remove TBS-T buffer and incubate membrane in the primary antibody solution (4 ul anti-His6 antibody in 20 ml 5% milk) for 24 hours at 4 oC. 9. Rinse the membrane 3 times for 10 min with TBS-T. 10. Incubate membrane in the secondary antibody solution (5ul Goat anti-mouse IgG in 20 ml 5% milk) for 30 minutes at room temperature. 11. Rinse the membrane 3 times for 10 min with TBS-T. 12. Mix the Solution A and B at a 1:1 ratio to make PicoEPD western blot detection substrate working solution (1ml of working solution is sufficient). 13. Wet the blotting membrane and cover the blot with clear plastic wrap. 14. Expose the membrane to films in a dark room.  Assay30 July 2020Binding assay of scFv(Ras) and HRas  1. Sample preparation    1. Buffer: 20 mM Tris-HCl pH 7.5 + 250 mM NaCl    2. scFv(Ras) sample: scFv(Ras) 250 ㎕ + buffer 250 ㎕    3. HRas G12V sample: HRas G12V 250 ㎕ + buffer 250 ㎕    4. scFv(Ras) + HRas G12V sample: scFv(Ras) 250 ㎕ + HRas G12V 250 ㎕    5. size exclusion chromatography (filtered buffer: 20 mM Tris-HCl pH 7.5 + 250 mM NaCl) 2. Equilibration: buffer 1CV 3. Sample loading: after equilibration, inject the sample 4. Elution: add 1 CV of buffer and collect 0.5 mL fractions using capless tube   ![](https://static.igem.org/mediawiki/2020/1/11/T--Korea_HS--lab6_1.png) |

2020 By iGEM team : Korea\_HS / e-mail : igem.koreahs@gmail.com
Copyrights(c) 2020 KORHS(KOREA\_HS) ALL RIGHTS RESERVED.

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

# Team:Korea HS/Results

# [KOREA\_HS](https://2020.igem.org/Team%3AKorea_HS)

[JUDGING](https://igem.org/2020_Judging_Form?team=Korea_HS)

* [TEAM](https://2020.igem.org/Team%3AKorea_HS/Team)

  + [Team](https://2020.igem.org/Team%3AKorea_HS/Team)
  + [Attributions](https://2020.igem.org/Team%3AKorea_HS/Attributions)
  + [Collaborations](https://2020.igem.org/Team%3AKorea_HS/Collaborations)
  + [Inclusion](https://2020.igem.org/Team%3AKorea_HS/Inclusion)
* [PROJECT](https://2020.igem.org/Team%3AKorea_HS/Contribution)

  + [Contribution](https://2020.igem.org/Team%3AKorea_HS/Contribution)
  + [Description](https://2020.igem.org/Team%3AKorea_HS/Description)
  + [Design](https://2020.igem.org/Team%3AKorea_HS/Design)
  + [Engineering](https://2020.igem.org/Team%3AKorea_HS/Engineering)
  + [Experiments](https://2020.igem.org/Team%3AKorea_HS/Experiments)
  + [Notebook](https://2020.igem.org/Team%3AKorea_HS/Notebook)
  + [Partnership](https://2020.igem.org/Team%3AKorea_HS/Partnership)
  + [Results](https://2020.igem.org/Team%3AKorea_HS/Results)
  + [Implementation](https://2020.igem.org/Team%3AKorea_HS/Implementation)
* [PARTS](https://2020.igem.org/Team%3AKorea_HS/Part_Collection)

  + [Part Collection](https://2020.igem.org/Team%3AKorea_HS/Part_Collection)
  + [Parts](https://2020.igem.org/Team%3AKorea_HS/Parts)
* [SAFETY](https://2020.igem.org/Team%3AKorea_HS/Safety)

  + [Safety](https://2020.igem.org/Team%3AKorea_HS/Safety)
* [HUMAN PRACTICES](https://2020.igem.org/Team%3AKorea_HS/Human_Practices)

  + [Human Practices](https://2020.igem.org/Team%3AKorea_HS/Human_Practices)
* [AWARDS](https://2020.igem.org/Team%3AKorea_HS/Education)

  + [Education](https://2020.igem.org/Team%3AKorea_HS/Education)
  + [Model](https://2020.igem.org/Team%3AKorea_HS/Model)
  + [Medal Criteria](https://2020.igem.org/Team%3AKorea_HS/Medal_Criteria)

## KOREA\_HS

Designing a Hyperstable Antibody with
Cell-penetrating Peptide for Intracellular Targeting

* [Contribution](https://2020.igem.org/Team%3AKorea_HS/Contribution)|  |
* [Description](https://2020.igem.org/Team%3AKorea_HS/Description)|  |
* [Design](https://2020.igem.org/Team%3AKorea_HS/Design)|  |
* [Engineering](https://2020.igem.org/Team%3AKorea_HS/Engineering)|  |
* [Experiments](https://2020.igem.org/Team%3AKorea_HS/Experiments)|  |
* [Notebook](https://2020.igem.org/Team%3AKorea_HS/Notebook)|  |
* [Partnership](https://2020.igem.org/Team%3AKorea_HS/Partnership)|  |
* [Results](https://2020.igem.org/Team%3AKorea_HS/Results)|  |
* [Implementation](https://2020.igem.org/Team%3AKorea_HS/Implementation)|  |

## Results

|  |
| --- |
| 1st Lab ResultsPCR amplification of insert (HRas G12V)  A. PCR of HRas ![](https://static.igem.org/mediawiki/2020/5/58/T--Korea_HS--rs1.png) Our team has amplified HRas using PCR and cloned the gene into an expression vector.    B. PCR of scFv(Ras) ![](https://static.igem.org/mediawiki/2020/b/b8/T--Korea_HS--rs1_1.png) Our team has amplified scFv(Ras) using PCR and cloned the gene into an expression vector.  2nd Lab ResultsTransformation and recombination of the plasmid (LIC vector - scFv(Ras)) to E. coli competent cell (BL21(DE)3) ![](https://static.igem.org/mediawiki/2020/2/22/T--Korea_HS--rs2.png)  Above is a plate obtained from transformation.  3rd & 4th Lab ResultsSDS-PAGE of purified histidine-tagged (scFv(Ras))  HRas (G12V) test expression ![](https://static.igem.org/mediawiki/2020/2/21/T--Korea_HS--rs3.png) We have tested protein expression in 4 different conditions (2 temperatures X 2 IPTG concentrations). After expression, the Ni-NTA column was used for protein purification using the His6-tag located at the N-terminus of CPP-scFv(Ras).     ![](https://static.igem.org/mediawiki/2020/7/75/T--Korea_HS--rs4.png) We have tested protein expression in 4 different conditions (2 temperatures X 2 IPTG concentrations). After expression, the Ni-NTA column was used for protein purification using the His6-tag located at the N-terminus of CPP-scFv(Ras).  5th Lab ResultsWestern blotting  A. CPP-scFv(Ras) western blot using anti-His6 antibody ![](https://static.igem.org/mediawiki/2020/6/64/T--Korea_HS--rs5.png) We have confirmed the identity of the protein using Western blot analysis using anti-His6 antibody.    B. HRas(G12V) western blot using anti-His6 antibody ![](https://static.igem.org/mediawiki/2020/1/10/T--Korea_HS--rs5_1.png) To confirm the identity of the protein, we performed Western blot using anti-his antibody. The result showed our protein and positive control(pc) is detected.  AssayBinding assay using size-exclusion column   ![](https://static.igem.org/mediawiki/2020/f/f5/T--Korea_HS--rs6.png) Using a size-exclusion column, we tested if scFv(Ras) and HRas forms a complex. From comparisons of three runs (scFv only, HRas only and scFv÷HRas), shift of HRas peak to higher molecular weight is observed. The complex formation was also confirmed by SDS~PAGE analysis.  Summary We were successful in:   * Amplifying HRas (G12V) and scFv(Ras) using PCR and cloning the gene into an expression vector. * Testing protein expression in 4 different conditions * Confirming the identity of the protein using Western blot analysis using anti-His6 antibody * Confirming the complex formation using size-exclusion chromatography and SDS-PAGE analysis     Unfortunately, we were not successful in:   * His6 tag cutting using TEV protease. SDS-Page results were unclear and therefore we had to send it for N-term sequencing |

2020 By iGEM team : Korea\_HS / e-mail : igem.koreahs@gmail.com
Copyrights(c) 2020 KORHS(KOREA\_HS) ALL RIGHTS RESERVED.