iGEM 2023 — Team ccu-taiwan

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
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![Notebook page!](  https://static.igem.wiki/teams/4674/wiki/notebook-header.png)

May.
Jun.
Jul.
Aug.
Sep.
Oct.

Week 1
Week 2
Week 3
Week 4
Week 5

Week 6
Week 7
Week 8
Week 9

Week 10
Week 11
Week 12
Week 13

Week 14
Week 15
Week 16
Week 17
Week 18

Week 19
Week 20
Week 21
Week 22

Week 23
Week 24

## Week 1 (4/30-5/6)

---

#### Wet Lab

* Discussing the necessity of DNA tetrahedron in the whole project.
* Exploring methods to demonstrate the success of DNA tetrahedron assembly.
* Identifying a peptide that can effectively label circulating tumor cells (CTCs) from references.
* Learning the principles of instruments used for observing DNA tetrahedron, including Atomic Force Microscope (AFM) and TEM.
* Discussing if PEG linker is necessary for the Folic acid conjugated single-stranded DNA.

#### Dry Lab

* Ensuring that team members are familiar with the assignment of tasks in the hardware process.
* Explaining the principle of Ficoll separation of peripheral blood myeloid cells (PBMC).
* Determining the type of pump and valves for Circulating Tumor Cells (CTC) detection.
* Discussing the storage conditions for fluorescent protein and DNAse.
* Draw the prototype of the Main Chamber.
* Micro channel design brainstorming - for separating CTCs.
* Quantitative Counting Method for fluorescence protein labeled CTCs.
* Exploring the principles and applications of Fluorescence Microscopy and Flow Cytometry.
* Animating CTC-FAST device model and its principles.
* CTC-FAST device prototype.
* Calculating the probability of CTC occurrence and the number of macrophages.
* Exploring methods to separate CTCs from macrophages
* Calculating the minimum volume of the Main Chamber.

#### Human Practice

* Brainstorming for CTC-FAST product promotion.
* Conducting the SWOT analysis for CTC-FAST product.
* After discussing with Professor Hong Jing, we decided to address the “Uneven Distribution of Medical Resources in Rural Areas" as the motivational direction.
* Collecting data on medical resources distribution in Taiwan.
* Collecting data on the medical challenges faced by rural patients in Taiwan.

#### Wiki

* Selecting color schemes and designing the concept for the Wiki.

## Week 2 (5/7-5/13)

---

#### Wet Lab

* Learning to use AFM.
* Inquiring about PEG-related questions with Dr. Tseng.
* Exploring whether mGreen Lantern (mGL) fluorescent protein affects the binding affinity of the CTC-recognining peptide.
* Inquiring with Dr. Tseng (with Biochemistry expertise) the pros and cons of PEG linker.
* Inquiring with Dr. Tsai about the modeling of mGL and CTC-recognining peptide fusion protein.
* Inquiring with Dr. Kan if we could use the AFM for DNA tetrahedron detection.
* Learning how to culture cells.

#### Dry Lab

* Estimating the probability of two CTCs being connected together.
* Discussing the calculation of flow rate, the probability algorithm, and the calculations involved in main chamber modeling.

#### Human Practice

* Collecting policies related to healthcare in Taiwan rural areas.
* Discussing the benefits that CTC-FAST can bring to society.
* Brainstorming future lung cancer prevention and awareness events that we can hold.

#### Wiki

* Designing the menu, footer parts of Wiki.

## Week 3 (5/14-5/20)

---

#### Wet Lab

* Discussing how to integrate our project into the society.
* Learning how to culture cells.
* Organizing the project description and the questions for consulting.

#### Dry Lab

* Estimating the cost of CTC-FAST device.
* Finding papers on pressure differences caused by microchannel junctions, and the feasibility of microfluidics and Raman spectroscopy

#### Human Practice

* Inviting the Taiwan Cancer Foundation to co-organize World Lung Cancer Day event.

#### Wiki

* Planning the open lab activities for educating high school students in synthetic biology.
* Scheduling future social media post themes and dates.

## Week 4 (5/21-5/2)

---

#### Wet Lab

* Learning how the pET vectors work.
* Learning the mechanism of Gibson assembly.
* Organizing how the pET-15b vector works.
* Discussing the safety form

#### Dry Lab

* Purchasing the materials of CTC-FAST device.
* Completing the Summer Personnel Schedule.

#### Human Practice

* Designing the first version of the board game for lung cancer education.
* Discussing the necessity of CTC-FAST detectors for rural areas.

#### Wiki

* Designing the appearance of the members page.
* Brainstorming the post-crossing activity.

## Week 5 (5/28-6/3)

---

#### Wet Lab

* Planning the experiment of DNA tetrahedron assembly.
* Preparing a Glossary for team members without biology expertise.

#### Dry Lab

* Learning 3D Printing machines and Electronics circuitry.
* Cold welding the peristaltic pump for pushing buffer in CTC-FAST.
* Applying stepper motor to control syringe injection in CTC-FAST.

#### Human Practice

* Listing experts who specialize in research related to healthcare in rural areas, and getting ready to interview them.
* Discussing and uploading the project description.

#### Wiki

* Illustrating the portraits of members (Leaders).
* Design the style of postcards.

## Week 6 (6/4~6/10)

---

#### Wet Lab

* Designing the ssDNA sequence for DNA tetrahedron.

#### Dry Lab

* Assembling the stepper motors onto the syringe.
* Photodiode device assembly.
* 3D printing of valves and laser placement.
* Designing light intensification and filtering device assembly.
* Measurement of Voltage and current value.

#### Wiki

* Designing and Coding parts page.
* Designing the appearance of Hardware pages of Wiki.

## Week 7 (6/11~6/17)

---

#### Wet Lab

* Organizing the experimental results and write notebooks.
* Performing the DNA tetrahedron assembly experiments with one, two, three or four ssDNAs in assembly buffer with 5mM Mg2+. Results examined by agarose gel electrophoresis.

#### Human Practice

* After discussing with the supervisor again, We thought that the original motivation to solve the difficulty of seeking medical treatment in rural areas could not be achieved. Therefore, another meeting was held to discuss a new motivation.

#### Wiki

* Designing the appearance of home page of Wiki.

## Week 8 (6/18~6/24)

---

#### Wet Lab

* Performing DNA tetrahedron assembly experiments again and change the concentration of Mg2+.
* Linearizing pET-15b by for restriction enzyme for mGL+peptide or EGFP+peptide protein production.
* Performing Gibson assembly to assemble mGL+peptide or EGFP+peptide expressing biobrick into linearized pET-15b.
* Performing colony PCR to pick the right clone for sequencing confirmation.

#### Dry Lab

* Basic training for microchannel

#### Human Practice

* Recruitment of openlab apprentices.
* Revising and Uploading Description
* Discussing the feasibility of incorporating a CTC detection device in the healthcare system with Professor Hong Jing.
* Search of Taiwan's National Health Insurance meeting records from 2013 to 2022 and compile favorable conditions for adding CTC-FAST to the insurance.
* Collecting global and local statistics about lung cancer death rates.

#### Wiki

* Illustrating the portraits of members (Human Practice).

## Week 9 (6/25~7/1)

---

#### Wet Lab

* Fusion Protein (mGL/eGFP+peptide) induction and SDS-PAGE confirmation.
* Optimizing DNA tetrahedron assembly.

#### Dry Lab

* Manufacturing steering engine (examine propulsion and flow control testing).
* Designing device for fluorescent detection (light path correction, examination photodiode, data analysis).

#### Human Practice

* Presenting a proposal to the Taiwan Cancer Society for the World Lung Cancer Day event plan.
* Making an on-campus fundraising presentation.
* Planning fundraising schedule.

#### Wiki

* Coding description page.
* Designing and Coding Notebook page.

## Week 10 (7/2-7/8)

---

#### Wet Lab

* Optimization of the first-generation DNA tetrahedron.
* mGreen Lantern protein expression and purification (small scale).
* Cloning the zinc finger binding protein by Gibson Assembly (pET-15b-PBSII+Zif268).
* Initiating the construction of long tetrahedral DNA (pET32a-ssDNA-L-R).
* SDS-PAGE confirmation of mGreen Lantern protein expression.
* Conjugating folic acid with the NHS.
* SDS-PAGE reconfirmation of mGreen Lantern protein expression.
* Harvesting the NHS-conjugated folic acid after agitating overnight, and purifying by filtration.

#### Dry Lab

* The assembly of the microcontroller-controlled pumping device.
* Adjustion for precise control of the outflow rate and time using the microcontroller-controlled pump (with an allowable error range of ±1ml / ±1s).
* The first version of the peristaltic pump power driving device.
* Successfully achievement of micro-dosing of liquids with one drop per output using the microcontroller-controlled pump.

#### Human Practice

* Surveying the place for Lung Cancer Day lecture and circulation planning.
* Completing documents related to on-campus fundraising (including proposals, speeches and presentations).
* Confirmation of World Lung Cancer Day venues.
* Consulting with previous team members about their experience in organizing events.
* Replan future work based on progress meeting feedback.

#### Wiki

* Surveying the place for Lung Cancer Day lecture and circulation planning.
* Completing documents related to on-campus fundraising (including proposals, speeches and presentations).
* Organizing Open Lab trial teaching activities.
* Designing the appearance of Human Practices.

## Week 11 (7/9-7/15)

---

#### Wet Lab

* Small-scale purification of mGreen Lantern.
* Continue cloning the zinc finger binding protein by Gibson Assembly (pET-15b-PBSII+Zif268).
* Sequencing the pET-15b-PBSII+Zif268.
* Construction of the long tetrahedral DNA (pET32a-ssDNA-L-R).
* Discussing the result of NHS-folic acid with Dr. Tseng and revised the protocol.
* Activating folic acid again.
* Harvesting the NHS-conjugated folic acid after agitating overnight, and purifying by filtration.

#### Dry Lab

* Outsourced the production of the Main chamber to a manufacturer.
* Completing the second version of the peristaltic pump power drive device.

#### Human Practice

* Reviewing and Revising the Lung Cancer Day activity plan and flow chart.
* Creating a venue map for the World Lung Cancer Day event.

#### Wiki

* Editing the contents of notebook (May) and coding the Notebook page.
* Preparing for the trial run of the open lab event.

## Week 12 (7/16-7/22)

---

#### Wet Lab

* SDS-PAGE confirmation of zinc finger binding protein (PBSII+Zif268) expression.
* Conjugating folic acid with the NHS again.
* SDS-PAGE confirmation of mGreen Lantern protein expression.
* Ligation gene blocks of the long tetrahedral DNA (ssDNA-L-R) by ligation PCR.
* Cloning the ligated gene block (ssDNA-L-R) into pET32a.
* Gel electrophoresis confirming pET32a-ssDNA-L-R by restriction enzyme digestion.
* Sequencing confirmation of pET32a-ssDNA-L-R.
* mGreen Lantern protein purification (small scale).
* Zinc finger binding protein (PBSII+Zif268) expression and purification (small scale).
* SDS-PAGE confirmation of mGreen Lantern protein purification.

#### Dry Lab

* Drawing the mold of trident microchannel by Solidwork. The main channel is with a diameter 500 micrometers, and the branch channel is with a diameter 300 micrometers.
* Printing and solidifying the mold of trident microchannel by light-curing 3D printer and ultraviolet light, respectively.
* Pulling the degased 10:1 mixture of ethylene benzene and PMS into mold to generate trident microchannel.
* Placing the mixture filled mold on an electromagnetic heating stirrer, baking it for 4 hours at 85C.
* Bonding the trident microchannel to glass slide and subjecting them to ozone for 40 minutes.
* Successfully let the Main Chamber shake by a servo motor.

#### Human Practice

* Confirming the speakers with the Taiwan Cancer Foundation for World Lung Cancer Day.
* Preparing a budget for the World Lung Cancer Day event.
* Confirming the list of staff members for the World Lung Cancer Day event.
* Arrangement of on-campus fundraising personnel.

#### Wiki

* Editing the contents of notebook (June) and coding the Notebook page.
* Conducting a trial run for the Open Lab event (7/17).

## Week 13 (7/23-7/29)

---

#### Wet Lab

* Analyzing the NHS-folic acid by Thin Layer Chromatography (TLC).
* Cloning the long tetrahedral DNA by Gibson Assembly (pET32a-ssDNA-L-R).
* Learning large scale purification of mGreen Lantern.
* Analyze the NHS-folic acid by Fourier-transform infrared spectroscopy (FTIR).
* Gel electrophoresis confirmation pET32a-ssDNA-L-R after restriction enzyme digestion.
* Searching the method for crosslinking PBSII+Zif268 to PMMA.

#### Dry Lab

* Improving the skill for trident microchannel production, and generating trident microchannels with main channel of 300 micrometers in diameter and branch channel of 100 micrometers.
* Completing the integrated propulsion system to connect with the Main Chamber.
* Circuit Amplification Issues and Discussion.

#### Human Practice

* Sending interview invitations to lung cancer specialists and clinics offering CTC testing.
* Interviewing Reporter Qiu-Ping Gao from the Formosa Cancer Foundation.
* Interviewing Dr. Gi-Ming Lai, the executive director of the Formosa Cancer Foundation, to discuss the experience of lung cancer prevention.
* Introducing iGEM competetion in an Instagram post.
* Check the draft of on-campus fundraising personnel.
* Calling the medical laboratory to invite them for an interview.

#### Wiki

* Coding Human Practices page.
* Contacting the manufacturer to order team uniforms and postcards.
* Conducting final trial run for Open Lab. (7/24)
* Holding the Open Lab event. (7/29)

## Week 14 (7/30-8/5)

---

#### Wet Lab

* Discussing the FA-conjugated DNA purification methods with Dr. Tseng and protocol revison.
* Cloning the long tetrahedral DNA by Gibson Assembly (pET32a-ssDNA-L-R).
* Gel electrophoresis confirming pET32a-ssDNA-L-R by restriction enzyme digestion.
* SDS-PAGE confirmation of zinc finger binding protein (PBSII+Zif268) expression.
* SDS-PAGE confirmation of mGreenLantern protein expression.
* Large-scale purification of mGreen Lantern.
* Discussing the dialysis formulation with Dr. Tseng and protocol revision.

#### Dry Lab

* Discussing how to build the liquid propulsion device for the microchannel.
* Experiment proved that mGL can be detected by fluorescent detection under the concentration of 1.83\*10^-11 mg/ml.
* Improving the wire configuration and voltage issues of the liquid microcontroller.

#### Human Practice

* Introducing synthetic biology concepts on Instagram post.
* Venue survey for World Lung Cancer Day.
* Organizing World Lung Cancer Day event.

#### WIki

* Editing the contents of notebook (July) and coding the Notebook page.
* Editing description page.
* Holding the Open Lab event. (8/5)
* Contacting the Manufacturer to Order Team merchandising (stickers).

## Week 15 (8/6-8/12)

---

#### Wet Lab

* Initiating the construction of long tetrahedral DNA (pET15b-tetrahedral DNA-RCR).
* Cloning the long tetrahedral DNA by Gibson Assembly (pET15B-tetrahedral DNA-RCR).
* Gel electrophoresis confirming pET15b-tetrahedral DNA-RCR by restriction enzyme digestion.
* Cloning the long tetrahedral DNA by Gibson Assembly (pET15b-SSB-RepA).
* mGreen Lantern protein quantification.
* mGreen Lantern protein cryopreservation.
* First attempt at performing a DNA dialysis experiment.
* Harvesting the products of DNA dialysis.
* SDS-PAGE confirmation of the products of DNA dialysis.
* Second attempt at performing a DNA dialysis experiment.
* Harvesting the products of DNA dialysis.
* SDS-PAGE confirmation of the products of DNA dialysis.
* Re-transformation of the Zinc finger binding protein (PBSII+Zif268).
* SDS-PAGE confirmation of zinc finger binding protein (PBSII+Zif268) expression.

#### Dry Lab

* Building the liquid propulsion system of the microchannel.
* Detecting the voltage of excitation light generated by laser light from different concentrations of fluorescent substances.
* Applying AD620 amplifier to our fluorescent detection system and collect mGL data under different concentrations.

#### Human Practice

* Introducing the work that the CTC-FAST team was engaged in on Instagram.
* Fundraising in the College of Science and the College of Social Sciences.
* Contact medical laboratories, foundations, and lung cancer associations to invite for interviews, which plays a role in optimizing our product and providing constructive suggestions.

#### Wiki

* Illustrating the portraits of members (Wet Lab).
* Designing and Coding protocol and proof of concept page.

## Week 16 (8/13-8/19)

---

#### Wet Lab

* Ligation gene blocks of the long tetrahedral DNA (ssDNA-L-R) by ligation PCR.
* Cloning the long tetrahedral DNA by Gibson Assembly (pET32a-ssDNA-L-R).
* Gel electrophoresis confirmation pET32a-ssDNA-L-R after restriction enzyme digestion.
* Cloning the ligated gene block (ssDNA-L-R) into pET32a.
* Gel electrophoresis confirmation pET15b-SBP-RepA after restriction enzyme digestion.
* Sequencing confirmation of pET15b-SBP-RepA.
* Preservation of mGreen Lantern protein by freeze-drying.
* Fluorescence detection confirming the fluorescence intensity of mGreen Lantern.
* First attempt at performing a Folic acid conjugate to ssDNA experiment.
* Purify FA-ssDNA after dialysis.
* SDS-PAGE confirmation of Folic acid conjugate ssDNA (FA-ssDNA).
* Research papers on inclusion body solubilization.

#### Dry Lab

* Building the liquid propulsion system of the microchannel.
* Using the amplifier module to analyze the data of unpurified mGL.

#### Human Practice

* Hold a video interview with Dr. Huang to gain insights into the current development and Application of CTC detection.
* Drafted the interview outline and sent interview invitations to the Taiwan Society of Laboratory Medicine.

#### Wiki

* Editing the content of attributions, filling the attribution form, and coding attribution page.
* Designing and Coding Hardware page.

## Week 17 (8/20-8/26)

---

#### Wet Lab

* Sequencing confirmation of pET15b-SSB-RepA.
* SDS-PAGE confirmation of pET15b-SSB-RepA expression.
* Learning cell culture.
* mGreen Lantern protein cryopreservation.
* Large-scale purification of mGreen Lantern.
* Fluorescence detection confirming the fluorescence intensity of mGreen Lantern.
* SDS-PAGE confirmation of mGreen Lantern protein expression.
* Cultivating SKOV3 cells for CTC mimic.
* Second attempt at performing a Folic acid conjugate to ssDNA experiment.
* Analyzing the spectrum of the FA-ssDNA conjugate using NanoDrop.
* Collecting reference for inclusion body solubilization.

#### Dry Lab

* Improving the liquid propulsion system of the microchannel.
* Discussing the problem of liquid inflow into the microchannel and discuss the solution to it.
* Using the amplifier module to detect the data of purified mGL.

#### Human Practice

* Fundraising with the Management College.
* Interview Dr. Yao-Lin Tzeng, chairman of the Taiwan Association of Thoracic & Cardiovascular Surgery.
* Interviewing Dr. Chih-Ying Liao, founder of Cancell.
* Interviewing Dr. Gi-Ming Lai, executive director of the Formosa Cancer Foundation, to gain insights into the current development and Application of CTC detection.
* Interviewing Dr. Chia-Jui Yen, director of the National Cheng Kung University Hospital Cancer Center.

#### Wiki

* Illustrating the portraits of members (Dry Lab).
* Coding the Safety and Model pages.

## Week 18 (8/27-9/2)

---

#### Wet Lab

* Cloning the long tetrahedral DNA by Gibson Assembly (pET32a-ssDNA-L-R).
* Gel electrophoresis confirmation pET32a-ssDNA-L-R after restriction enzyme digestion.
* SDS-PAGE confirmation of pET15b-SSB-RepA expression.
* Analyzing the spectrum of the FA-ssDNA conjugate using NanoDrop.
* Cultivating SKOV3 cells as CTC mimics.
* Purification of inclusion bodies (pET-15b-PBSII+Zif268).

#### Dry Lab

* Generating a thicker microchannel with 4 mm thickness.
* Making thicker aluminum plates on the microfluidic liquid propulsion system to improve the stability of the propelled liquid.
* Trying to solve the liquid exudate problem of the microchannel.

#### Human Practice

* Conducting an interview with a lung cancer patient.
* Interview with the case manager.
* Interview Dr. Mei-Chia Wang, chief Medical Technologist at Chang Gung Hospital.
* Interview Mr. Tsai, Manager at LifeCode Biotech.

#### Wiki

* Designing the appearance of sponsors, and coding Sponsors page.
* Designing and coding Implementation page.

## Week 19 (9/3-9/9)

---

#### Wet Lab

* Cloning the long tetrahedral DNA by Gibson Assembly (pET32a-ssDNA-L-R).
* Gel electrophoresis confirmation pET32a-ssDNA-L-R after restriction enzyme digestion.
* Purification of inclusion bodies (pET-15b-PBSII+Zif268).
* Learn to use oxygen plasma to modify NHS on PMMA.
* Research papers on refolding of solubilized protein (pET-15b-PBSII+Zif268).

#### Dry Lab

* Finish the Contribution and Hardware writeups for the Wiki.

#### Human Practice

* List of nutrient-rich foods and discussion with school cafeteria about collaborative ‘Healthy bento for healthy lungs’ nutrient lunch box programs.
* Fundraising event in Chiayi temple.
* Promotion video shooting.
* Planning short, medium, and long-term goals for FAST CTC.

#### Wiki

* Editing the contents of notebook (August) and coding the Notebook page.
* Designing and coding Contribution page.

## Week 20 (9/10-9/16)

---

#### Wet Lab

* Ligation gene blocks of the long tetrahedral DNA (ssDNA-L-R) by ligation PCR.
* Cloning the long tetrahedral DNA by Gibson Assembly (pET32a-ssDNA-L-R).
* Gel electrophoresis confirmation pET32a-ssDNA-L-R after restriction enzyme digestion.
* Cloning the ligated gene block (ssDNA-L-R) into pET32a.
* Fourth attempt at performing a Folic acid conjugate to ssDNA experiment.
* Purify FA-ssDNA after dialysis.
* Analyze the spectrum of the FA-ssDNA conjugate using NanoDrop.
* Treating PMMA with oxygen plasma.
* Modifying PMMA with NHS.
* Subjecting the activated folic acid to UV-vis spectroscopy.
* Learning safranin staining.
* Staining PMMA with Safranin O.
* Cultivating SKOV3 cells to simulate CTCs.
* Purification of inclusion bodies (pET-15b-PBSII+Zif268).

#### Dry Lab

* Drawing CTC-FAST's internal 3D modeling design for Contribution.

#### Human Practice

* Inquire about ISO certification.
* Compile and summarizing interview records.
* Contact a specialist from the Legal Affairs Department.

#### Wiki

* Illustrating the portraits of members (Investigators and Instructors).
* Coding Material page.

## Week 21 (9/17-9/23)

---

#### Wet Lab

* Cloning the long tetrahedral DNA by Gibson Assembly (pET32a-ssDNA-L-R).
* Gel electrophoresis confirmation pET32a-ssDNA-L-R after restriction enzyme digestion.
* Treating PMMA with oxygen plasma.
* Modifying PMMA with NHS.
* Staining PMMA with Safranin O.
* Subjecting the PMMA to UV-vis spectroscopy.

#### Dry Lab

* Assembling the final overall CTC-FAST device.
* Making a video showing how CTC-FAST works.

#### Human Practice

* Composing Wiki education content.
* Composing Wiki outreach content.
* Composing Wiki Integrated Human Practice content.

#### Wiki

* Illustrating the portraits of members (Wiki and Advisors).
* Editing the content of Mempers and coding the Members page.

## Week 22 (9/24-9/30)

---

#### Wet Lab

* Cloning the long tetrahedral DNA by Gibson Assembly (pET32a-ssDNA-L-R).
* Gel electrophoresis confirmation pET32a-ssDNA-L-R after restriction enzyme digestion.
* Treating PMMA with oxygen plasma.
* Modifying PMMA with amino functional groups.
* Staining PMMA with Safranin O.
* Subjecting the PMMA to UV-vis spectroscopy.
* Conjugating PMMA with mGL to verify the successful NHS modification on the PMMA surface.

#### Human Practice

* Editing and uploading IHP, Education, Outreach Wiki content

#### Wiki

* Editing the contents of notebook (September) and coding the Notebook page.

## Week 23 (10/1-10/7)

---

#### Wiki

* Coding the Results and Judging pages.
* Check and modify the content and typesetting on wiki.

## Week 24 (10/8-10/12)

---

#### Wiki

* Editing the contents of notebook (October) and coding the Notebook page.
* Final check the content and typesetting on wiki.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering{% endblock %}
{% block lead %}Describe all the safety issues of your project.{% endblock %}
{% block page\_content %}

![Engineering page!](https://static.igem.wiki/teams/4674/wiki/engineering-header.png)

* [**Overview**](#Overview)
* [**DNA tetrahedron**](#DNA tetrahedron)
* [**mGL-C7 fusion protein**](#mGL-C7 fusion protein)
* [**References**](#References)

## **Overview**

---

The goal of CTC-FAST is to detect lung cancer metastasis. We achieve the capture of lung cancer CTCs by specifically targeting the folate receptor on the CTC membrane using folic acid. However, the CCU-TAIWAN team's objective goes beyond detecting lung cancer metastasis; we aim to create a biological machine that can detect metastasis in all types of cancers. To accomplish this, we utilize DNA nanostructures, DNA tetrahedron, to anchor folic acid in our machine. This allows us to easily replace the ligand in the future when we need to detect metastasis in other types of cancers. To create such a biological machine, we overcome several issues and improve our design by multiple engineering cycles as outlined below.

## **DNA tetrahedron**

---

#### -Round I- Tetrahedron from separated ssDNAs

**Design:**

We designed four separated single-strand DNAs (ssDNAs), which partially complement each other [(see design for details)](https://2023.igem.wiki/ccu-taiwan/design). The annealing of these four ssDNA will produce a DNA tetrahedron through base pairing.

* **TD1:** ACATTCCTAAGTCTGAAacATTACAGCTTGCTACACgaGAAGAGCCGCCATAGTA
* **TD2:** TATCACCAGGCAGTTGAcaGTGTAGCAAGCTGTAATagATGCGAGGGTCCAATAC
* **TD3:** TCAACTGCCTGGTGATAaaACGACACTACGTGGGAAtcTACTATGGCGGCTCTTC
* **TD4:** TTCAGACTTAGGAATGTgcTTCCCACGTAGTGTCGTttGTATTGGACCCTCGCAT
* **TD2F (TD2 with overhang for adaptor ssDNA recognition):** GCTTGCACGCGTGCtattaatATCACCAGGCAGTTGAcaGTGTAGCAAGCTGTAATagATGCGAGGGTCCAATAC

![](https://static.igem.wiki/teams/4674/wiki/engineering-td-okik.png)

**Build:**

We mixed four ssDNAs strands in TM buffer (50 mM Tris HCl, 10 mM Magnesium Sulfate at pH 7.5), heated the sample to 95°C for 5 mins, and gradually cooled the sample to 4°C over 2.5 hours.

**Test:**

We analyzed the mixed sample by native PAGE electrophoresis after gradually cooling. The mixture of 2 ssDNAs shows a size of 80 bp, while the mixture of 3 ssDNAs shows a size of 120 bp. Importantly, the mixture of 4 ssDNAs PAGE showed the expected size of 175 bp. However, we also observed side products with larger sizes, which may represent DNA polyhedrons, forming through complementation between tetrahedrons.

![](https://static.igem.wiki/teams/4674/wiki/2023-09-17-8-td-2.png)

**Learn:**

Due to the unexpected side product of DNA polyhedrons, we decided to change the design of 4 ssDNAs to one single tetrahedral ssDNA.

#### -Round II- Tetrahedron from single ssDNA

**Design:**

We decided to fuse the four separated ssDNA into one continuous ssDNA, namely tetrahedral ssDNA, and amplify it using in vitro rolling circle amplification (RCA). To cleave the long ssDNA into tetrahedral ssDNA, we incorporated cis-auto splicing sequences at the 3’ terminus of the tetrahedral ssDNA sequence.

![](https://static.igem.wiki/teams/4674/wiki/gene-2-engineering-design-2.png)

**Build:**

The production of tetrahedral ssDNA with highly complementary sequences was rejected by the manufacturer. Therefore, we separated the tetrahedral ssDNA into two fragments (ssDNA-L and ssDNA-R) and added an extra stuffer to reduce the complementarity.

After getting the two parts of tetrahedral ssDNA, we performed the fusion PCR to fuse these two fragments into one tetrahedral ssDNA with a stuffer. To remove the stuffer, we cloned the fused fragment into pET-32a vector, then removed the stuffer by EcoRI. Finally, we excised the tetrahedral ssDNA from pET-32a by XhoI, and self-ligated the excised tetrahedral ssDNA into a circular DNA template for RCA.

![](https://static.igem.wiki/teams/4674/wiki/gene-2-engineering-built.png)

**Test:**

The fusion PCR is successful, we could fuse the two fragment into one ssDNA with stuffer

![](https://static.igem.wiki/teams/4674/wiki/gene-2-test-2.png)

▲ The electrogram shows the sequencing result of ssDNA-L+R sequence

**Learn:**

Unfortunately, we are suggested by experts that the high concentration of DNA tetrahedrons generated by RCA also form polyhedrons. Therefore, we decided to apply ssDNA binding protein and in vivo rolling circle replication (RCR) for ssDNA production.

#### -Round III- Tetrahedron from a single ssDNA with ssDNA BP

**Design:**

To enhance DNA tetrahedron production, we applied RCR and expressed the ssDNA binding protein (SSBP) to protect the generated circular tetrahedral ssDNA. The SSBP is also applied to purify the binding ssDNA, as well as avoid unexpected interactions among ssDNAs.

**Build:**

To build the RCR system, we expressed the replication initiation proteins (RepA) from pC194 in *E. coli*. The tetrahedral ssDNA with cis-auto splicing sequence was flanked by the start motif (RCORI105) and stop motif (RCORI65) of RepA. The SSBP is co-expressed with Rep A to protect the tetrahedral ssDNA generated by RCR.

![](https://static.igem.wiki/teams/4674/wiki/gen3.png)

**Test:**

The fusion PCR is successful, we could fuse the two fragment into one ssDNA with stuffer.

![](https://static.igem.wiki/teams/4674/wiki/1.png)

▲ The electrogram shows the sequencing result of RcoRI-105 + ssDNA sequence + RcoRI-65

![](https://static.igem.wiki/teams/4674/wiki/2.png)

▲ The electrogram shows the Sanger sequencing result of ssDNA Binding protein - RepA

The RepA and SBP protein induction was confirmed by SDS-PAGE and Coomassie blue staining.

![](https://static.igem.wiki/teams/4674/wiki/sbp-repa-expression-okik.png)

▲ RepA and ssDNA binding protein (SBP) expression

## **mGL-C7 fusion protein**

---

**Design:**

To specifically label lung cancer CTCs, we fused mGreenLantern (mGL) protein with a 12-mer peptide (namely C7) specifically recognizing the membranous FRα.To ensure that C7 recognizes CTCs, we must keep the flexibility of C7 in mGL-C7 fusion protein. Based on previous studies, a linker composed of multiple Alanine residues was chosen. To determine the proper length of the linker, we decided to model the protein structure of mGL-C7 fusion protein with different linker lengths and simulate the docking of these fusion proteins on FRα.

**Build:**

We downloaded the PDB file of the FRα receptor and generated the mGL-C7 fusion protein with different lengths (0-4) of Alanine linker by I-TASSER (Zhou et al.).

**Test:**

To determine the proper length of the linker, we use Frodock (Ramírez-Aportela et. al, 2016 ) to perform molecular docking simulation between mGL-C7 with different linker lengths (0-4 Alanine) and the FRα.

Frodock mechanism: The Frodock website combines 3D grid-based potentials with the efficiency of spherical harmonics (SH) approximations for docking model simulation.

![](https://static.igem.wiki/teams/4674/wiki/docking-3d.png)

**Learn:**

Through observing the simulated docking results of fusion proteins with different linker lengths and FRα, we found that mGL-4A-C7 did not affect the docking of C7 on the FRα receptor and maintained the optimal distance between mGL and FRα. We then designed the expressing biobrick of mGL-4A-C7 and examined whether mGL-4A-C7 protein is functional.

|  | Top view | side view |
| --- | --- | --- |
| mGL-C7 | ![mGL-C7 Top view](https://static.igem.wiki/teams/4674/wiki/10-mgl-c7-topview.png) | ![mGL-C7 Side view](https://static.igem.wiki/teams/4674/wiki/9-mgl-c7-side-view.png) |
| mGL-1A-C7 | ![mGL-1A-C7 Top view](https://static.igem.wiki/teams/4674/wiki/2-mgl-1a-c7-topview.png) | ![mGL-1A-C7 Side view](https://static.igem.wiki/teams/4674/wiki/1-mgl-1a-c7-side-view.png) |
| mGL-2A-C7 | ![mGL-2A-C7 Top view](https://static.igem.wiki/teams/4674/wiki/4-mgl-2a-c7-topview.png) | ![mGL-2A-C7 Side view](https://static.igem.wiki/teams/4674/wiki/3-mgl-2a-c7-side-view.png) |
| mGL-3A-C7 | ![mGL-3A-C7 Top view](https://static.igem.wiki/teams/4674/wiki/6-mgl-3a-c7-top-view.png) | ![mGL-3A-C7 Side view](https://static.igem.wiki/teams/4674/wiki/5-mgl-3a-c7-side-view.png) |
| mGL-4A-C7 | ![mGL-4A-C7 Top view](https://static.igem.wiki/teams/4674/wiki/8-mgl-4a-c7-top-view.png) | ![mGL-4A-C7 Side view](https://static.igem.wiki/teams/4674/wiki/7-mgl-4a-c7-side-view.png) |

**Test:**

The coding sequence expressing mGL-4A-C7 was cloned into pET15b expressing vector by Gibson Assembly, and the expressed protein was analyzed by WB. After purifying the mGL-4A-C7 by Ni-sepharose beads and FPLC, we treated the SKOV3 cells with mGL-4A-C7 to examine whether the fusion protein was functional.

|  | control | 50 μg/mL | 100 μg/mL | 200 μg/mL |
| --- | --- | --- | --- | --- |
| nucleus | ![ Incubate 2hr nucleus-control bottom](https://static.igem.wiki/teams/4674/wiki/2-0mgl-b-bottom.jpg) | ![Incubate 2hr nucleus-50 bottom](https://static.igem.wiki/teams/4674/wiki/2-50mgl-b-bottom.jpg) | ![Incubate 2hr nucleus-100 bottom](https://static.igem.wiki/teams/4674/wiki/2-100mgl-b-bottom.jpg) | ![Incubate 2hr nucleus-200 bottom](https://static.igem.wiki/teams/4674/wiki/2-200mgl-b-bottom.jpg) |
| mGL-4A-C7 | ![Incubate 2hr  eGFP-4A-C7-control bottom](https://static.igem.wiki/teams/4674/wiki/2-0mgl-g-bottom.jpg) | ![Incubate 2hr eGFP-4A-C7-50 bottom](https://static.igem.wiki/teams/4674/wiki/2-50mgl-g-bottom.jpg) | ![Incubate 2hr  eGFP-4A-C7-100 bottom](https://static.igem.wiki/teams/4674/wiki/2-100mgl-g-bottom.jpg) | ![Incubate 2hr eGFP-4A-C7-200 bottom](https://static.igem.wiki/teams/4674/wiki/2-200mgl-g-bottom.jpg) |
| Merge | ![Incubate 2hr merage-control bottom](https://static.igem.wiki/teams/4674/wiki/2-0mgl-m-bottom.jpg) | ![Incubate 2hr merage-50 bottom](https://static.igem.wiki/teams/4674/wiki/2-50mgl-m-bottom.jpg) | ![Incubate 2hr merage-100 bottom](https://static.igem.wiki/teams/4674/wiki/2-100mgl-m-bottom.jpg) | ![Incubate 2hr merage-200 bottom](https://static.igem.wiki/teams/4674/wiki/2-200mgl-m-bottom.jpg) |

## **References**

---

X Zhou, W Zheng, Y Li, R Pearce, C Zhang, EW Bell, G Zhang, Y Zhang. I-TASSER-MTD: a deep-learning-based platform for multi-domain protein structure and function prediction. Nature Protocols, 17: 2326-2353 (2022)

E. Ramírez-Aportela, J.R. López-Blanco, and P. Chacón (2016). FRODOCK 2.0: Fast Protein-Protein docking server. Bioinformatics, 32(15), 2386-2388

{% endblock %}


{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

![Results page!](https://static.igem.wiki/teams/4674/wiki/results-header.png)

* [**Overview**](#Overview)
* [**The assembly of DNA tetrahedron by four separated ssDNA**](#The assembly of DNA tetrahedron by four separated ssDNA )
* [**The production of single tetrahedral ssDNA**](#The production of single tetrahedral ssDNA)
* [**The construction of RCR system**](#The construction of RCR system)
* [**The folic acid (FA) conjugated adaptor ssDNA**](#The folic acid (FA) conjugated adaptor ssDNA)
* [**The expression of PBSII-Zif268 fusion protein**](#The expression of PBSII-Zif268 fusion protein)
* [**The functional confirmation of mGL-4A-C7 proteins**](#The functional confirmation of mGL-4A-C7 proteins)

## **Overview**

---

In CTC-FAST project, we aim to improve the detection of circulating tumor cells (CTCs) by replacing the antibody based detection assay with DNA based one. Accordingly, we designed the
**folic acid (FA) conjugated DNA tetrahedrons** to capture CTCs from the blood sample. To anchor the DNA tetrahedrons in our automatic device, we decided to apply the
**zinc finger fusion protein Zif268-PBSII**, which will be coated on the main chamber of our device to interact with the corresponding motifs at the edge of DNA tetrahedron. To produce the tetrahedral ssDNA with highly complementary sequences in vivo, we construct the
**rolling circle replication (RCR)** system in E. coli. Finally, to label the captured CTCs, we used the new fluorescence protein, **mGreenLatern (mGL)**, which has a stronger brightness than canonical eGFP, to increase the sensitivity.

## **The assembly of DNA tetrahedron by four separated ssDNA (success)**

---

To examine whether the DNA tetrahedron could be assembled by single-stranded DNA (ssDNA) in a complementary manner, we designed four ssDNAs TD-1, TD-2, TD-3 and TD-4
(Sequences are shown in [design](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D)) and performed the tetrahedron assembly according to the [**standard annealing protocol**](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D).

After annealing, the reaction mixture was subjected into native PAGE electrophoresis. The product size of annealing two ssDNAs (TD-1+TD-2 and TD-2+TD-3) was around 80 bp, while annealing three ssDNAs (TD-1+TD-3+TD-4 and TD-2+TD-3+TD-4) was around 120 bp. The product sizes of annealing two or three ssDNA were slightly smaller than anticipation, which may result from the partial complementation. Importantly, the product size of annealing four ssDNAs was 175 bp, suggesting a more completely complementation. However, we also observed the side product with size more than 175 bp, which may stand for the DNA polyhedrons.

![Fig. 1: The native PAGE analysis of annealing ssDNAs following the standard protocol.](https://static.igem.wiki/teams/4674/wiki/fig-1-result-okik.png)

▲ Fig. 1: The native PAGE analysis of annealing ssDNAs following the standard protocol.

Because the Mg2+ is important for DNA complementation, we speculated whether the increase of Mg2+ concentration would inhibit the unexpected polyhedron assembly. Accordingly, we increased the Mg2+ concentration from 5 mM to 25 mM. However, the native PAGE analysis showed no significant improvement of tetrahedron assembly.

![Fig. 2: The native PAGE analysis of annealing ssDNAs with different Mg2+ concentration.](https://static.igem.wiki/teams/4674/wiki/fig-2-result-okik.png)

▲ Fig. 2: The native PAGE analysis of annealing ssDNAs with different Mg2+ concentration.

Since the standard protocol cool the ssDNA mixture fastly, we guess this may cause unexpected interaction. Therefore, we try to gradually decrease the annealing temperature (3~4 °C/5 min), allowing the tetrahedron to properly assemble. However, the native PAGE analysis still shows no improvement significantly.

![Fig. 3:The native PAGE analysis of tetrahedron assembly by gradually cooling down.                   ](https://static.igem.wiki/teams/4674/wiki/fig-3-result-okik.png)

▲ Fig. 3:The native PAGE analysis of tetrahedron assembly by gradually cooling down.

Together, we thought that the tetrahedron assembly from separated ssDNA may form side products like polyhedrons, which could be caused by the random complementation among the ssDNAs.

## **The production of single tetrahedral ssDNA (success)**

---

To rescue the random complementation, we decided to fuse the four ssDNAs into single tetrahedral ssDNA for tetrahedron assembly. Because of the reverse polarity of complementary DNA strands, one edge of the tetrahedron must form the “twin double helices” structure
(See [**design**](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D) for detail). We decided to generate the double-stranded DNA (dsDNA) encoding tetrahedral sequence as a template and performed in vitro rolling circle amplification (RCA) to produce the tetrahedral ssDNA. However, the serious problem is that the production of dsDNA templates with highly complementary sequences are rejected by most companies. To put the tetrahedral dsDNA template into order, we separated the dsDNA template into two parts (ssDNA-L and ssDNA-R) and added an extra stuffer to reduce the complementarity.

![Fig. 4: The drawing shows how we add stuffer and separate tetrahedral ssDNA to reduce the complementarity.](https://static.igem.wiki/teams/4674/wiki/fig-4-result-okik.png)

▲ Fig. 4: The drawing shows how we add stuffer and separate tetrahedral ssDNA to reduce the complementarity.

After getting the two parts of tetrahedral ssDNA (ssDNA-L and ssDNA-R), we performed the fusion PCR to fuse these two fragments into one tetrahedral ssDNA with a stuffer. The TA ligation and sequencing confirmed the inserted fragment was indeed the fusion of ssDNA-L and ssDNA-R.

![Fig. 5: The electrogram shows the sequence of fused ssDNA-L and ssDNA-R](https://static.igem.wiki/teams/4674/wiki/fig-5-result-okik.png)

▲ Fig. 5: The electrogram shows the sequence of fused ssDNA-L and ssDNA-R

#### -Engineering

The experiment went smoothly. We could remove the stuffer by EcoRI digestion and self-ligation. The dsDNA template for tetrahedral ssDNA could be excised form TA vector and relegated as RCA template. However, after discussing with the experts, we realized that the single tetrahedral ssDNA may also cause polyhedron side products.

## **The construction of RCR system to express tetrahedral ssDNA (On going)**

---

According to the suggestion from experts, we decided to construct the RCR system in E.coli to generate circular tetrahedral ssDNAs from the dsDNA template and coexpress ssDNA binding protein (SSBP) to block the polyhedrons formation.

![Fig. 6: The design of RCR mediated circular ssDNA generation](https://static.igem.wiki/teams/4674/wiki/fig-7-drsign-okik.png)

▲ Fig. 6: The design of RCR mediated circular ssDNA generation

Accordingly, we builded two composite parts. One composite part
([BBa\_K4674011](http://parts.igem.org/Part%3ABBa_K46740011))
is applied to express the RCR initiation proteins (RepA) and SSBP and the other one
([BBa\_K4674012](http://parts.igem.org/Part%3ABBa_K4674012))
is the target dsDNA template cassette for RepA recognition
(see [design](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D) for detail). After cloning, the sequencing result confirmed that two composite parts were successfully cloned into the pET-15b expression vector.

![Fig. 7: The electrogram shows the successful insertion of target cassette ](https://static.igem.wiki/teams/4674/wiki/fig-6-result-okik.png)

▲ Fig. 7: The electrogram shows the successful insertion of target cassette
([BBa\_K4674012](http://parts.igem.org/Part%3ABBa_K4674012))

![Fig. 8: The electrogram shows the successful insertion of RepA and SSBP expression cassette](https://static.igem.wiki/teams/4674/wiki/fig-7-result-okik.png)

▲ Fig. 8: The electrogram shows the successful insertion of RepA and SSBP expression cassette
([BBa\_K4674011](http://parts.igem.org/Part%3ABBa_K46740011))

We then induced the RepA and SSBP protein expression by IPTG. After 6 hours of IPTG induction at 37°C, we found that the RepA and SSBP were expressed. However, the expression of protein was found in the inclusion body (cell pellet).

![Fig. 9: The coomassie blue staining of SDS-PAGE analysis of RepA and SSBP protein induction. S: supernatant; P:  cell pellet.               ](https://static.igem.wiki/teams/4674/wiki/fig-8-result-okik.png)

▲ Fig. 9: The coomassie blue staining of SDS-PAGE analysis of RepA and SSBP protein induction. S: supernatant; P: cell pellet.

#### -Future work

To confirm whether the RCR system is functional, we will perform the protein induction at lower temperature (e.g. 12~16°C), and examine the protein expression by SDS-PAGE analysis. After successfully inducing the protein expression, we will purify the his-tagged SSBP by Ni2+ beads, and perform PCR analysis to examine whether the RCR system successfully produces the circular tetrahedral ssDNA from the target cassette. Finally, the cis-autosplicing of purified circular tetrahedral ssDNA will be induced by Mg2+ ion, and the formation of tetrahedron will be examined by atomic force microscope (AFM).

## **The folic acid (FA) conjugated adaptor ssDNA (success)**

---

To conjugate the folic acid to the adaptor ssDNA for CTC capture, we utilized the mechanism of SN2 reaction to prepare FA-NHS ester. Following the
[**protocol**](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocol%27%29%20%7D%7D) of generating FA-NHS ester, we observed the expected pale yellow substance.

![Fig. 10: The folic acid NHS ester shows pale yellow feature.](https://static.igem.wiki/teams/4674/wiki/fig-9-result-okik.jpg)

▲ Fig. 10: The folic acid NHS ester shows pale yellow feature.

To confirm its identity, we utilized Fourier-transform infrared spectroscopy (FTIR) for qualitative analysis of the substance. The IR spectrum of FA-NHS ester indicated that the main component is FA, as compared to the standard IR spectrum of FA provided by spectral Database for Organic Compounds (SDBS). Importantly, three characteristic peaks (2700 ~ 3300 cm-1) in the IR spectrum of FA-NHS are also shown in the standard IR spectrum of NHS. The slight shift of wavelength of these peaks may be caused by the conjugation. Together, this result suggested the successful conjugation of NHS to FA.

![Fig. 11: The folic acid NHS ester IR spectra compare with SDBS ](https://static.igem.wiki/teams/4674/wiki/fig-10-result-okik.png)

▲ Fig. 11: The folic acid NHS ester IR spectra compare with SDBS

Subsequently, we reacted the FA-NHS ester with NH2 modified adaptor ssDNA, resulting in the formation of FA-ssDNA. Since the FA compound show absorption around the 300 nm, we performed UV-vis spectroscopy to observe the absorption spectra, showing that the absorption spectra of FA-ssDNA and ssDNA almost completely overlap, with the former showing a slight elongation of the peak at approximately 305 nm.

![Fig.12: The absorption spectra of FA-ssDNA and ssDNA ](https://static.igem.wiki/teams/4674/wiki/fig-11-result-okik.png)

▲ Fig.12: The absorption spectra of FA-ssDNA and ssDNA

## **The expression of PBSII-Zif268 fusion protein: (On going)**

---

To anchor the DNA tetrahedrons in our automatic device, we applied the specific interaction between zinc finger fusion protein (PBSII-Zif268) and its corresponding motifs at the edge of the tetrahedron
(see [**design**](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D)for details). To express the PBSII-Zif268 fusion protein, we design the composite part
 and clone it into pET-15b plasmid. The sequencing result confirmed the correct insertion of
[BBa\_K4674013](http://parts.igem.org/Part%3ABBa_K4674013) in the pET-15b vector.

![Fig. 13:The electrogram shows the successful insertion of PBSII-Zif268 expression cassette.                 ](https://static.igem.wiki/teams/4674/wiki/fig-13-result-okik.png)

▲ Fig. 13:The electrogram shows the successful insertion of PBSII-Zif268 expression cassette.

After confirming the cloning, we induced the PBSII-Zif268 fusion protein expression by treating 0.25 mM IPTG at 37°C. The PAGE analysis showed that the fusion protein was located in the inclusion body (P lane). We then modified the induction temperature to 16°C, however, the PAGE analysis result still showed the inclusion body location of PBSII-Zif268 fusion protein.

![Fig. 14: The coomassie blue staining of SDS-PAGE analysis of PBSII-Zif268 fusion protein induced at different temperatures and IPTG concentrations.  Sup: supernatant; P: pellet                  ](https://static.igem.wiki/teams/4674/wiki/fig-14-result-okik.png)

▲ Fig. 14: The coomassie blue staining of SDS-PAGE analysis of PBSII-Zif268 fusion protein induced at different temperatures and IPTG concentrations. Sup: supernatant; P: pellet

We then try to extract the PBSII-Zif268 fusion protein from the inclusion body by 8 M Urea solution. The PAGE analysis showed that the treatment of 8 M Urea could solubilize the PBSII-Zif268 fusion protein (the Sup 2 lane).

![Fig. 15: The coomassie blue staining of SDS-PAGE analysis of PBSII-Zif268 fusion protein extracted from the inclusion body. Sup 2: the urea-soluble supernatant; P2: the urea-insoluble pellet ](https://static.igem.wiki/teams/4674/wiki/fig-15-result-okik.png)

▲ Fig. 15: The coomassie blue staining of SDS-PAGE analysis of PBSII-Zif268 fusion protein extracted from the inclusion body. Sup 2: the urea-soluble supernatant; P2: the urea-insoluble pellet

#### -Future work

The 8 M Urea extracted PBSII-Zif268 fusion protein needs to be purified by Ni2+ bead and renature into the functional structure for docking DNA tetrahedron. We have found the protocol for protein renature on the Ni2+ beads or after elution. The corresponding experiments will be performed to optimize the PBSII-Zif268 fusion protein collection.

## **The functional confirmation of mGL-4A-C7 proteins (success)**

---

To examine whether the mGL-4A-C7 protein is functional in labeling CTCs as the modeling result, we builded the composite part
([BBa\_K4674010](http://parts.igem.org/Part%3ABBa_K4674010))
for mGL-4A-C7 protein expression. As a control, we also builded the composite part
([BBa\_K4674009](http://parts.igem.org/Part%3ABBa_K4674009)) for eGFP-4A-C7 protein expression.

After confirming the expression cassette insertion into pET-15b, we induced the mGL-4A-C7 and eGFP-4A-C7 protein expression by adding 1mM IPTG at 37°C. The Coomassie blue staining of PAGE analysis clearly showed that the induced mGL-4A-C7 and eGFP-4A-C7 protein was soluble and could be purified by Ni2+ beads.

![Fig. 16: The coomassie blue staining of SDS-PAGE analysis of mGL-4A-C7 and eGFP-4A-C7 protein induced by IPTG.                       ](https://static.igem.wiki/teams/4674/wiki/fig-16-result-okik-1-new.png)

![Fig. 16: The coomassie blue staining of SDS-PAGE analysis of mGL-4A-C7 and eGFP-4A-C7 protein induced by IPTG.                       ](https://static.igem.wiki/teams/4674/wiki/fig-16-result-okik-2.png)

▲ Fig. 16: The coomassie blue staining of SDS-PAGE analysis of mGL-4A-C7 and eGFP-4A-C7 protein induced by IPTG.

To purify a large batch of mGL-4A-C7 and eGFP-4A-C7 protein for further examination and application, we performed the FPLC analysis to separate the non-specific binding protein from target protein. The absorption spectra at 280 nm indicated that there are two main proteins containing fractions. One is from 14th to 18th fraction, and the other is from 21st to 38th fractions.

![Fig. 17: The 280 nm absorption spectra of proteins eluted from mGL-4A-C7 purification.](https://static.igem.wiki/teams/4674/wiki/fig-17-result-okik.png)

▲ Fig. 17: The 280 nm absorption spectra of proteins eluted from mGL-4A-C7 purification.

Although the greens fluorescence clearly shown in the 21-38 fraction, we still performed the SDS-PAGE analysis, confirming the existence of mGL-4A-C7 protein.

![Fig. 18: The coomassie blue staining of SDS-PAGE analysis of fractions from FPLC elution.](https://static.igem.wiki/teams/4674/wiki/fig-18-result-okik-1.png)
![Fig. 18: The coomassie blue staining of SDS-PAGE analysis of fractions from FPLC elution.](https://static.igem.wiki/teams/4674/wiki/fig-18-result-okik-2.png )

▲ Fig. 18: The coomassie blue staining of SDS-PAGE analysis of fractions from FPLC elution.

After confirming the existence of target proteins, we freeze-dried the fractions containing mGL-4A-C7 or eGFP-4A-C7 proteins. The freeze drying protein was then rehydrated to the same concentration for examination.

We first examine whether the mGL exhibits a strong brightness when excited by 488 nm laser. The value of fluorescence brightness detected by standard eGFP filter indicated that, the mGL exhibits fluorescence at least twofold fluorescence brighter than eGFP at the same concentration, and protein could be stored at room temperature at least one week.

![Fig. 19: The fluorescence detection of rehydrated mGL-4A-C7 and eGFP-4A-C7 stored at room temperature using the standard eGFP filter. ](https://static.igem.wiki/teams/4674/wiki/fig-19-result-okik.png)

▲ Fig. 19: The fluorescence detection of rehydrated mGL-4A-C7 and eGFP-4A-C7 stored at room temperature using the standard eGFP filter.

Next, we examine whether the mGL-4A-C7 protein could recognize the CTC mimics, the SKOV3 cells. Accordingly, we incubated SKOV3 cells with medium containing different concentrations of mGL-4A-C7 and eGFP-4A-C7 (50μg/ml, 100μg/ml, 200μg/ml) at 37 ℃ for 2 hours. The observation of fluorescence indicated that the 50 μg/mL of mGL-4A-C7 could label the SKOV3 cells, while the 50 μg/mL of eGFP-4A-C7 showed moderate labeling. Furthermore, the extension of incubating time to 4 hours did not significantly improve the labeling. Together, we confirmed that the mGL-4A-C7 could label the SKOV3 cells, suggesting mGL-4A-C7 protein as CTC labeling reagent.

| Incubate 2hr | | | | |
| --- | --- | --- | --- | --- |
|  | control | 50 μg/mL | 100 μg/mL | 200 μg/mL |
| nucleus | ![ Incubate 2hr nucleus-control top](https://static.igem.wiki/teams/4674/wiki/2-0gfp-b-top.jpg) | ![Incubate 2hr nucleus-50 top](https://static.igem.wiki/teams/4674/wiki/2-50gfp-b-top.jpg) | ![Incubate 2hr nucleus-100 top](https://static.igem.wiki/teams/4674/wiki/2-100gfp-b-top.jpg) | ![Incubate 2hr nucleus-200 top](https://static.igem.wiki/teams/4674/wiki/2-200gfp-b-top.jpg) |
| eGFP-4A-C7 | ![Incubate 2hr  eGFP-4A-C7-control top](https://static.igem.wiki/teams/4674/wiki/2-0gfp-g-top.jpg) | ![Incubate 2hr eGFP-4A-C7-50 top](https://static.igem.wiki/teams/4674/wiki/2-50gfp-g-top.jpg) | ![Incubate 2hr  eGFP-4A-C7-100 top](https://static.igem.wiki/teams/4674/wiki/2-100gfp-g-top.jpg) | ![Incubate 2hr eGFP-4A-C7-200 top](https://static.igem.wiki/teams/4674/wiki/2-200gfp-g-top.jpg) |
| Merge | ![Incubate 2hr merage-control top](https://static.igem.wiki/teams/4674/wiki/2-0gfp-m-top.jpg) | ![Incubate 2hr merage-50 top](https://static.igem.wiki/teams/4674/wiki/2-50gfp-m-top.jpg) | ![Incubate 2hr merage-100 top](https://static.igem.wiki/teams/4674/wiki/2-100gfp-m-top.jpg) | ![Incubate 2hr merage-200 top](https://static.igem.wiki/teams/4674/wiki/2-200gfp-m-top.jpg) |
|  | control | 50 μg/mL | 100 μg/mL | 200 μg/mL |
| nucleus | ![ Incubate 2hr nucleus-control bottom](https://static.igem.wiki/teams/4674/wiki/2-0mgl-b-bottom.jpg) | ![Incubate 2hr nucleus-50 bottom](https://static.igem.wiki/teams/4674/wiki/2-50mgl-b-bottom.jpg) | ![Incubate 2hr nucleus-100 bottom](https://static.igem.wiki/teams/4674/wiki/2-100mgl-b-bottom.jpg) | ![Incubate 2hr nucleus-200 bottom](https://static.igem.wiki/teams/4674/wiki/2-200mgl-b-bottom.jpg) |
| mGL-4A-C7 | ![Incubate 2hr  eGFP-4A-C7-control bottom](https://static.igem.wiki/teams/4674/wiki/2-0mgl-g-bottom.jpg) | ![Incubate 2hr eGFP-4A-C7-50 bottom](https://static.igem.wiki/teams/4674/wiki/2-50mgl-g-bottom.jpg) | ![Incubate 2hr  eGFP-4A-C7-100 bottom](https://static.igem.wiki/teams/4674/wiki/2-100mgl-g-bottom.jpg) | ![Incubate 2hr eGFP-4A-C7-200 bottom](https://static.igem.wiki/teams/4674/wiki/2-200mgl-g-bottom.jpg) |
| Merge | ![Incubate 2hr merage-control bottom](https://static.igem.wiki/teams/4674/wiki/2-0mgl-m-bottom.jpg) | ![Incubate 2hr merage-50 bottom](https://static.igem.wiki/teams/4674/wiki/2-50mgl-m-bottom.jpg) | ![Incubate 2hr merage-100 bottom](https://static.igem.wiki/teams/4674/wiki/2-100mgl-m-bottom.jpg) | ![Incubate 2hr merage-200 bottom](https://static.igem.wiki/teams/4674/wiki/2-200mgl-m-bottom.jpg) |

| Incubate 4hr | | | | |
| --- | --- | --- | --- | --- |
|  | control | 50 μg/mL | 100 μg/mL | 200 μg/mL |
| nucleus | ![ Incubate 4hr nucleus-control top](https://static.igem.wiki/teams/4674/wiki/4-0gfp-b-top.jpg) | ![Incubate 4hr nucleus-50 top](https://static.igem.wiki/teams/4674/wiki/4-50gfp-b-top.jpg) | ![Incubate 4hr nucleus-100 top](https://static.igem.wiki/teams/4674/wiki/4-100gfp-b-top.jpg) | ![Incubate 4hr nucleus-200 top](https://static.igem.wiki/teams/4674/wiki/4-200gfp-b-top.jpg) |
| eGFP-4A-C7 | ![Incubate 4hr  eGFP-4A-C7-control top](https://static.igem.wiki/teams/4674/wiki/4-0gfp-g-top.jpg) | ![Incubate 4hr eGFP-4A-C7-50 top](https://static.igem.wiki/teams/4674/wiki/4-50gfp-g-top.jpg) | ![Incubate 4hr  eGFP-4A-C7-100 top](https://static.igem.wiki/teams/4674/wiki/4-100gfp-g-top.jpg) | ![Incubate 4hr eGFP-4A-C7-200 top](https://static.igem.wiki/teams/4674/wiki/4-200gfp-g-top.jpg) |
| Merge | ![Incubate 4hr merage-control top](https://static.igem.wiki/teams/4674/wiki/4-0gfp-m-top.jpg) | ![Incubate 4hr merage-50 top](https://static.igem.wiki/teams/4674/wiki/4-50gfp-m-top.jpg) | ![Incubate 4hr merage-100 top](https://static.igem.wiki/teams/4674/wiki/4-100gfp-m-top.jpg) | ![Incubate 4hr merage-200 top](https://static.igem.wiki/teams/4674/wiki/4-200gfp-m-top.jpg) |
|  | control | 50 μg/mL | 100 μg/mL | 200 μg/mL |
| nucleus | ![ Incubate 4hr nucleus-control bottom](https://static.igem.wiki/teams/4674/wiki/4-0mgl-b-bottom.jpg) | ![Incubate 4hr nucleus-50 bottom](https://static.igem.wiki/teams/4674/wiki/4-50mgl-b-bottom.jpg) | ![Incubate 4hr nucleus-100 bottom](https://static.igem.wiki/teams/4674/wiki/4-100mgl-b-bottom.jpg) | ![Incubate 4hr nucleus-200 bottom](https://static.igem.wiki/teams/4674/wiki/4-200mgl-b-bottom.jpg) |
| mGL-4A-C7 | ![Incubate 4hr  eGFP-4A-C7-control bottom](https://static.igem.wiki/teams/4674/wiki/4-0mgl-g-bottom.jpg) | ![Incubate 4hr eGFP-4A-C7-50 bottom](https://static.igem.wiki/teams/4674/wiki/4-50mgl-g-bottom.jpg) | ![Incubate 4hr  eGFP-4A-C7-100 bottom](https://static.igem.wiki/teams/4674/wiki/4-100mgl-g-bottom.jpg) | ![Incubate 4hr eGFP-4A-C7-200 bottom](https://static.igem.wiki/teams/4674/wiki/4-200mgl-g-bottom.jpg) |
| Merge | ![Incubate 4hr merage-control bottom](https://static.igem.wiki/teams/4674/wiki/4-0mgl-m-bottom.jpg) | ![Incubate 4hr merage-50 bottom](https://static.igem.wiki/teams/4674/wiki/4-50mgl-m-bottom.jpg) | ![Incubate 4hr merage-100 bottom](https://static.igem.wiki/teams/4674/wiki/4-100mgl-m-bottom.jpg) | ![Incubate 4hr merage-200 bottom](https://static.igem.wiki/teams/4674/wiki/4-200mgl-m-bottom.jpg) |

{% endblock %}