A Novel Reconstruction and Expression of Endostatin with pDC316 Plasmid in Eukaryotic Cells

Student: Ziqi Wang
Table: MED1215
Experimentation location: Reseach Institution
Regulated Research (Form 1c): No
Project continuation (Form 7): No

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Abstract:

Endostatin is one of the strongest endothelial cell inhibitory factors that has been found so far, however, endostatin protein is extremely unstable and difficult to prepare and apply. The goal of this research is to investigate a novel biotechnology method to reconstruct endostatin. Subcloning endostatin gene into pDC316 plasmid and transfect into human embryonic kidney 293T cells is proposed in this research.

Endostatin gene is amplified by polymerase chain reaction (PCR). Purified endostatin gene fragments were ligated to eukaryotic expression vector pDC316. The reconstructed plasmids were transformed into competent cells E. coli DH5α. Single and clear-edged bacteria are tested by bacterial PCR and electrophoresis. Plasmid was extracted from the positive bacteria and was sequenced for further confirmation. The endostatin containing plasmid was transfected into 293T cells, the expression of the target gene in the infected cells was detected by reverse transcription-polymerase chain reaction(RT-PCR) and gel electrophoresis.

The final product reconstructed by endostatin DNA with the pDC316 vector was confirmed by sequencing and endostatin mRNA was successfully expressed in transfected 293T cells. This research successfully reconstructed endostatin into pDC316 plasmid vector and transfected the identified plasmid into 293T cells. This recombinant endostatin has potential value angiogenesis gene therapy.

Keywords
Recombinant Endostatin, Angiogenesis, pDC316, 293T cells

Bibliography/Citations:

References

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Additional Project Information

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Research Plan:

A. Questions or Problem being addressed
Endostatin is one of the strongest endothelial cell inhibitory factors that has been found so far, however, endostatin protein is extremely unstable and difficult to prepare and apply. 
B. Goals/Expected Outcomes/Hypothesis
The goal of this research is to investigate a novel biotechnology method to reconstruct endostatin.
C. Methods/Procedures
Endostatin gene is amplified by polymerase chain reaction (PCR). Purified endostatin gene fragments were ligated to eukaryotic expression vector pDC316. The reconstructed plasmids were transformed into competent cells E. coli DH5α. Single and clear-edged bacteria are tested by bacterial PCR and electrophoresis. Plasmid was extracted from the positive bacteria and was sequenced for further confirmation. The endostatin containing plasmid was transfected into 293T cells, the expression of the target gene in the infected cells was detected by reverse transcription-polymerase chain reaction(RT-PCR) and gel electrophoresis. Endostatin gene is amplified by polymerase chain reaction (PCR). Purified endostatin gene fragments were ligated to eukaryotic expression vector pDC316. The reconstructed plasmids were transformed into competent cells E. coli DH5α. Single and clear-edged bacteria are tested by bacterial PCR and electrophoresis. Plasmid was extracted from the positive bacteria and was sequenced for further confirmation. The endostatin containing plasmid was transfected into 293T cells, the expression of the target gene in the infected cells was detected by reverse transcription-polymerase chain reaction(RT-PCR) and gel electrophoresis. The final product reconstructed by endostatin DNA with the pDC316 vector was confirmed by sequencing and check whether it will be successfully be expressed in transfected 293T cells.

 

 

 

 

Questions and Answers

1. What was the major objective of your project and what was your plan to achieve it? 

Since endostatin protein is extremely unstable and difficult to prepare and apply, the goal of this project is to investigate a novel technology method to reconstruct endostatin. 

       a. Was that goal the result of any specific situation, experience, or problem you encountered?  

Not really for my personal experience, but after reading articles, I found it really interesting to study. 

       b. Were you trying to solve a problem, answer a question, or test a hypothesis?

Solve a problem. 

2. What were the major tasks you had to perform in order to complete your project?

Multiple biogenetic techniques need to be performed in order to reconstruct endostatin.

3. What is new or novel about your project?

In this project, the goal is to investigate a novel technology method to reconstruct endostatin. 

       a. Is there some aspect of your project's objective, or how you achieved it that you haven't done before?

There are some biogenetic techniques that I have never used before. 

       b. Is your project's objective, or the way you implemented it, different from anything you have seen?

No. From my perspective, this project follows the same approach as many other studies. Start with a hypothesis, set your own plan and do experiments to prove it.

       c. If you believe your work to be unique in some way, what research have you done to confirm that it is?

Through a lot of reading of related articles, I found that no one had tried the method used in this study. 

4. What was the most challenging part of completing your project?

Because I have not been experienced in a large laboratory before, sometimes unskilled or unregulated operation may occur

      a. What problems did you encounter, and how did you overcome them?

Sometimes I wasn't sure if I was doing the right thing, so asked my mentor to get the answer

      b. What did you learn from overcoming these problems?

I learned how to do operations properly, but more importantly, I learned how to communicate better with my mentors and observed what a researcher's day is like.

5. If you were going to do this project again, are there any things you would you do differently the next time?

I should have started my research earlier so that I could get more in-depth results

6. Did working on this project give you any ideas for other projects? 

Yes, my interest in doing biological research has increased a lot

7. How did COVID-19 affect the completion of your project?

Due to COVID-19, I could not complete the experiments in the school lab, but had to go back to China to complete them, and I had to communicate with my teacher through online channels at times.