Engineering Success
Engineering success is based on the four stages of design, building, testing, and learning, which can help us explore and improve our chassis microbes.
1. The first generation:
1.1 Design
Our goal is for people who want to lose weight with our E. coli Nissle1917 designed to reduce weight efficiently and painlessly. We have designed E. coli Nissle 1917 and plan to place it in the intestine of patients. These E. coli have the ability to enhance the expression of long-chain fatty acid transport proteins, which can help people lose weight by taking up fatty acids in the intestine and slowing down from being absorbed by the gut.
1.2 Building
In E. coli Nissle1917, the unidirectional transport of long-chain fatty acids (LCFA) is dominated by two proteins, FadL (outer membrane fatty acid transporter protein), which provides a channel in the cell membrane for the ingestion of long-chain fatty acids by the body. Once the long-chain fatty acids enter the cells, they are metabolized by E. coli Nissle1917 and excreted in the feces.
1.3 Testing
| Control group (empty plasmid only) | FadL only | |
|---|---|---|
| Extracellular |
++++++++8 7.96 7.87 8.23/8.02 |
+++++5 4.97 4.86 5.35/5.06 |
| Endocellular |
++2 1.78 2.05 2.32/2.05 |
+++3 3.09 3.21 3.03/3.11 |
| Total |
++++++++++10 10.07 |
++++++++8 8.17 |
According to our experiments, we found that the extracellular concentration of palmitic acid decreased to nearly half when the strain contained the FadL gene compared to the control; while the intra-package concentration increased. This indicates that the purpose of our strain design, i.e., to accelerate the rate of palmitic acid transport by probiotics increased.
1.4 Learning
It is through the experimental results that although we increased the ability of the strain to absorb and transport fatty acids, its overall metabolic efficiency was not significantly improved. Based on this, we thought that we should also increase the metabolic ability of the strain after it absorbs fatty acids, so we tried to find a gene that enhances the efficiency of bacterial metabolism of fatty acids.
2. The second generation:
2.1 Design
We found that another FadL gene contributes to the uptake efficiency of our E. coli, and based on this, we redesigned a gene line.LCFA binds to FadL, and as the conformation changes, LCFA crosses the outer membrane through FadL. LCFA is protonated in the outer membrane space and then enters the inner membrane by diffusion.
2.2 Building
(less wiring diagram) In this part, we redesigned the system of E. coli 1917 metabolism by adding genes related to fadD in the plasmid. This can help boost the efficiency of our probiotic. Within the inner membrane, FadD activates LCFA to CoA thioester. After activation, LCFA undergoes β-oxidation to provide respiratory substrate.
2.3 Testing
We added the FadD gene to the strain, and according to the experimental data, we could see that when only the FadD gene was present in the strain, the extracellular concentration of palmitic acid did not change much, but the intracellular concentration dropped dramatically by three times, which indicated that the FadD gene played a role in degrading palmitic acid in our experiments. In contrast, when both FadL and FadD genes were expressed in the cells, both the extracellular and intracellular concentrations of palmitic acid decreased dramatically.
| Control group (empty plasmid only) | FadL only | FadD only | Both FadL FadD | |
|---|---|---|---|---|
| Extracellular |
++++++++8 7.96 7.87 8.23/8.02 |
+++++5 4.97 4.86 5.35/5.06 |
+++++++7 7.13 7.04 6.77/6.98 |
+++3 3.43 3.09 3.26/3.26 |
| Endocellular |
++2 1.78 2.05 2.32/2.05 |
+++3 3.09 3.21 3.03/3.11 |
+1 1.15 1.36 1.18/1.23 |
++2 1.99 1.74 1.82/1.85 |
| Total |
++++++++++10 10.07 |
++++++++8 8.17 |
++++++++8 8.21 |
+++++5 5.11 |
According to our experiments, we found that the extracellular concentration of palmitic acid decreased to nearly half when the strain contained the FadL gene compared to the control; while the intra-package concentration increased. This indicates that the purpose of our strain design, i.e., to accelerate the rate of palmitic acid transport by probiotics increased.
2.4 Learning
After comparison, we found that the strains that added the FadD gene and FadL had a significantly higher metabolic fatty acid efficiency than the characterization effect of the two single genes. Based on this, we believe that our experimental results are in line with the experimental expectations.
3. The third generation:
3.1 Design
We plan to use a biosafety system composed of L-Rhamnose, Rhamnose promoter PRha, Regulator AraC, Alanine racemase Alr, Terminator, Arabinose promoter PBAD, and RNase Ba. This is because although a large number of igem teams and experiments have proven that E. coli nissle1917 is almost harmless to the environment and organisms, we designed this suicide system to prevent the leakage of E. coli due to biosafety requirements and the requirements of the igem organizing committee. We designed a small molecule suicide system based on the controlled intake of rhamnose. Ultimately, we achieve the destruction of dextroalanine, while initiating the expression of downstream toxic protein Barnase, which initiates a double kill switch system and can basically ensure the death of E. coli nissle1917.
3.2 Building
Conditional-suicide containment system: We plan to use a biosafety system composed of L-Rhamnose, Rhamnose promoter PRha, Regulator AraC, Alanine racemase Alr, Terminator, Arabinose promoter PBAD, and RNase Ba. Combining the above genes with the biosafety strain K-12 ∆alr ∆dadX creates a powerful device that allows us to control the division of bacterial cells. The control of bacterial growth can be active or passive. Active is the induction of the PBAD promoter with L-arabinose and passive is the induction with L-rhamnose. Passive control makes it possible to control bacterial cell division in a defined closed environment, such as MFC, by continuously adding L-rhamnose to the culture medium. As shown on the left, this leads to the expression of the necessary alanine racemase (alr) and AraC regulators, resulting in the inhibition of RNase Ba expression.
If E. coli Nissle1917 leaves the defined environment of the MFC or if L-rhamnose is no longer added to the medium, the expression of both alanine racemase (Alr) and the AraC regulator is reduced and therefore the toxic RNase Ba (Barnase) starts to be expressed. the cleavage of intracellular RNA by Barnase and the D- caused by the inhibited alanine racemase The lack of alanine synthesis inhibits cell division and ensures that the bacteria can only grow in a defined environment or in a selected device. When the bacteria are excreted by the body, or when the body stops ingesting rhamnose, which will cause them die out.
Reference:
https://2013.igem.org/Team:Bielefeld-Germany/Biosafety/Biosafety_System_S#Biosafety-System_araCtive
3.3 Testing
During the 24-hour test cycle, we started adding rhamnose to the medium at hour 0 and stopped adding it at hour 9. During this time, the OD600 values of the control group and the test group, which could represent bacterium activity, were almost the same. However, after stopping the addition of rhamnose at the 9th hour, the activity of the test group decreased dramatically and eventually converged to 0
3.4 Learning
The suicide system worked properly, meeting our experimental expectations and proving that our bacteria do not leak and contaminate the environment.
Thus, with two more generations of construction tests and enhancements of the basic metabolic system, and one generation of suicide system design, we succeeded in cultivating the E. coli Nissle1917 we wanted and got the desired results.