Engineering Success | NJMU-China

This page shows the engineering iterations of our two systems and proves our engineering success. For DESIGN, through a process of questioning, we developed our design progressively. For BUILD, agarose gel electrophoresis was used to verify the successful construction and transformation of our plasmids. For TEST, we performed different experiments for each system and successfully verify their function. For LEARN, we provided conclusions of our successes, failures, and future plans for each system after iGEM.

System 1

Design

Wet Lab Design

What substance will we choose to degrade the brain autoantibody GAD?Why?

What genes will we select to synthesize the substance?

Which protein will we use to act as a induction switch?Why?

Which strategy will we choose to assure security? Why?

What chassis bacteria will we use? Why?

To answer the above question, we did literature reviews and brainstorming sessions to complete the design of system one. You can find more information on the Design page.

The map of system 1

Build

Wet Lab Build

We successfully constructed the pSIP403-N-acyltransferase-PnisA-nisRK-erY plasmid and verified it by agarose gel electrophoresis. You can find more information on the Results page.

Test

Wet Lab Test

We conducted molecular experiment and function verification. You can find more information on theResults page.

Dry Lab Test

You can find more information on the Modelpage.

Learn

Successe

We successfully transformed the pSIP403-N-acyltransferase-PnisA-nisRK-erY plasmid into E. coli DH5α and confirmed it through agarose gel electrophoresis.

Failure

However, due to plasmid design issues, upon transformation into Lactobacillus Plantarum L168, we observed homologous recombination within the plasmid through PCR and agarose gel electrophoresis, resulting in the appearance of two bands on the gel. Consequently, we have reflected upon the shortcomings in plasmid design and aim to make improvements in our next experiment.

Future Plans

Conduct the molecular docking experiment.
In the future, we hope to verify that ferrichrome can bind to other brain autoantibodies.

System 2

Design

Wet Lab Design

What substance will we choose to degrade the brain autoantibody MBP?Why?

What genes will we select to synthesize the substance?

Which protein will we use to act as a induction switch?Why?

Which strategy will we choose to assure security? Why?

What chassis bacteria will we use? Why?

To answer the above question, we did literature reviews and brainstorming sessions to complete the design of system two. You can find more information on the Design page.

The map of system 2

Build

Wet Lab Build

We successfully constructed the pSIP403-hemA-PnisA-nisRK-erY plasmid and verified it by agarose gel electrophoresis.

You can find more information on the Results page.

Test

Wet Lab Test

At the DNA level, we performed enzyme digestion/plasmid PCR and ran agarose gel to prove the successful construction or transformation of the plasmid.

In function verification,we drew the bacterial growth curve and verified their suiside switch function.

You can find more information on the Results page.

Dry Lab Test

You can find more information on the Model page.

Learn

Successes

We successfully transformed the pSIP403-N-acyltransferase-PnisA-nisRK-erY plasmid into E. coli DH5α and confirmed the transformation through agarose gel electrophoresis.
Furthermore, we successfully introduced the plasmid into Lactobacillus Plantarum L168 and verified the transformation through agarose gel electrophoresis.
Finally, we confirmed the upregulation of protoporphyrin transcription levels through qPCR.

Failures

At the outset, we encountered difficulties in successfully transforming L168. Subsequently, upon analysis, we identified the poor state of the recipient as the underlying issue. Consequently, we reprepared the recipient , ultimately achieving successful transformation.

Future plans

Conduct the molecular docking experiment.