Wet Lab Design
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What substance will we choose to enhance mitochondrial function? Why? |
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What role does it play in enhancing mitochondrial function? |
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Which genes will we select to directly or indirectly produce the substance? Why? How does it work? |
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Which strategy will we choose to assure security? Why? |
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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
Wet Lab Build
We successfully constructed the LDHLH673-PncA plasmid and verified it by agarose gel electrophoresis. You can find more information on the Results page.
Wet Lab Test
You can find more information on the Results page.
In our pre-experiments, we knocked out the PncA gene in Lactobacillus Plantarum L168 and found it would produce fewer NAD+, which provides indirect evidence to the test stage. You can find more information on the Results page.
In Silico Test
For the purpose of understanding the absorption and distribution of NAD+ in the human body, we developed a 3D visualization and two videos. The organ model files are from bodyparts 3D. The model was created using Blender, Python scripts, and Meshlab, and the video was rendered using Blender.
In the video, the transparent pink contour represents the human body, the red and pink organs represent the liver and intestine, the fluorescent green spheres represent NAD+, and the blue blood vessels represent veins.
Video 1 provides an overview of our 3D visualization, in which we can observe the distribution of NAD+ in the small intestine, liver, mesenteric vein, and hepatic portal vein.
Video 2 is the focused view of our three-dimensional visualization, in which we can observe NAD+ being absorbed in the small intestine, entering the mesenteric vein, following the blood into the hepatic portal vein, and then entering the liver.
This 3D representation allows us to intuitively comprehend the distribution and absorption of NAD+ in the body. Originally, it was part of our pharmacokinetic model, however, without sufficient experimental data or literature support, it was not able to solve and develop this model. But we still kept the video and hope to complete it after iGEM.
Successes
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Construct the pLDHLH673-PncA plasmid
Failures
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Amplify the pMG36e and pLDHLMCS plasmid
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Transform the pLDHLH673-PncA into Lactobacillus Plantarum L168
Possible reasons: First, the plasmid extraction kit we used was ineffective. In the lysing bacteria step, the color of the solution should have become clear after adding the reagent, but it remained unchanged. Second, the chassis bacteria we choose, Lactobacillus Plantarum, are gram-positive bacteria that have different physiological characteristics from the gram-negative bacteria commonly used in plasmid extraction.
Future Plans
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Verify the function of PncA directly
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Verify the function of the Nisin system.
Wet Lab Design
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What substance will we choose to prevent the accumulation of heavy metals in children with autism? Why? |
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How does it work and what role does it play in preventing the accumulation of heavy metals in children with autism? |
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What genes will we select to synthesize the substance? Need some improvement? |
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Which strategy will we choose to assure security? Why? |
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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
Wet Lab Build
At the DNA level, we performed enzyme digestion/plasmid PCR and ran agarose gel to prove the successful construction or transformation of the plasmid.
At the protein level, we use the heavy metal solution of different concentrations to induce the protein expression and ran SDS-PAGE gal to prove the successful expression of our target gene.
You can find more information on the Results page.
In Silico Build
We established an ordinary differential (ODE) model to simulate the induction-expression process in this system. You can find more information on the Model page.
Wet Lab Test
We drew the bacterial growth curve. We and verified their heavy metal elimination function and as well as kill switch function.
In Silico Test
Combined with experimental data, the induction-expression process in this system was visualized. You can find more information on the Model page.
Successes
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Transform the pET28a-MT plasmid into Escherichia coli Nissle 1917
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Verify the protein expression
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Draw the growth curve
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Verified the heavy metal elimination function
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Verify the kill switch function
Failures
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Determine whether the target protein is anchored to the bacterial outer membrane
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Determine The elimination capacity of our engineering bacteria to other heavy metal ions
Future plans
In the future, we hope to complete experiments on the sense and elimination capacity of engineered bacteria to different heavy metal ions (lead, mercury, copper, zinc, lithium), we hope to perform immunofluorescence staining that can determine the localization of the target protein in the engineered bacteria.
Wet Lab Design
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Why do we consider drug delivery? |
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What drug delivery methods are available and what are they? |
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What is the definition of microencapsulation and what is its function? |
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How can we achieve microencapsulation? |
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How can we validate the microencapsulation results? |
To answer the above question, we did literature reviews and brainstorming sessions to complete the design of system three. You can find more information on the Design page.
Web Lab Build
To confirm the establishment of our microencapsulation, electron microscopic images were taken. By counting the number of bacteria before and after microencapsulation, we also measured the microencapsulation yield. You can find more information on th Results page.
Illustration of microencapsulation
Wet Lab Test
We let our engineered bacteria with or without microencapsulation pass the simulating gastrointestinal fluid and then evaluate their survival rates to test if microencapsulation could help engineered bacteria survive the harsh environment of the human digestive tract. You can find more information on the Results page.
Illustration of microencapsulation verification
During the process of adding the suspension to the calcium chloride solution using a sterile nozzle syringe to form bacterial microcapsules, it was found that controlling the syringe at a certain height and injecting at a uniform speed resulted in more homogeneous microcapsules. No attention was paid to controlling the height and speed of injection during the first experiment and this was improved during the second experiment.
Successes
We have successfully produced microencapsulated engineered bacteria and confirmed its activity with functional validation.
Failures
We tried using yogurt as a vehicle for drug delivery, but the smear plate results showed that the Lactobacillus plantarum colony count was much higher than expected. This may have been caused by the bacteria continuing to proliferate during multiple failed vacuum drying attempts.
Future plans
In the future, we hope to complete the functional validation of microencapsulated engineered bacteria with adsorbed heavy metal ions.
Wet Lab Design
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What substances do we use as markers of mitochondrial dysfunction? Why? |
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What system do we choose to sense lactic acid? Why? How does it sense lactic acid? |
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What substances do we choose to report? Why? |
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Which strategy will we choose to assure security? Why? |
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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 four. You can find more information on the Design page.
The map of system 4
Wet Lab Build
At the DNA level, we performed enzyme colony/plasmid PCR and ran agarose gel to prove the successful construction or transformation of the plasmid.
At the protein level, we use the lactate of different concentrations to induce the protein expression and ran SDS-PAGE gal to prove the successful expression of our target gene.
You can find more information on the Results page.
In Silico Build
We established an ordinary differential (ODE) model to simulate the induction-expression process in this system. You can find more information on the Model page.
Wet Lab Test
We verified the lactate test function
You can find more information on the Results page.
In Silico Test
Combined with experimental data, the induction-expression process in this system was visualized. You can find more information on the Model page.
Successes
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Transform ALPaGA-lacZ into Escherichia coli DH5α-T1
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Verify that the engineered bacteria could produce more Beta-galactosidase under the increasing of lactate concentration.
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Prediction curve of lactate-protein expression
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On the test paper, the engineered bacteria produced color reaction with x-gal.
Failures
According to the conclusion that engineering bacteria can produce more Beta-galactosidase by a high concentration of lactic acid, we propose to distinguish different concentrations of lactate according to the degree of color reaction, so as to diagnose autism, however, only a little bit of difference was found in our functional test, which may be related to the material of filter paper, the lower activity of bacteria after fixing on the testing strip, and the unsuitable reaction environment.
Future plans
Further improve the sensitivity of the functional verification experiment.
