@@ -79,6 +79,21 @@ Fig1. Schematic model of Part1 system<br> If there is no IPTG TurboID will not b
As for our basic materials, biotin and ATP, we thought we could use engineered <i>Escherichia coli</i> (<i>E.coli</i>) to produce what we needed. Since there is already more than enough ATP in an organism, we only need to increase the production of biotin. We have two ways to achieve this. One is by increasing the production of biotin in one specific metabolic pathway, and the other is that we can block the other channel. However, blocking other channels will lead to a decrease in the growth rate and productivity of the bacteria, so we chose to increase the amount of the catalyst, which in turn produces more biotin. We will be increasing the catalyst of these two catalysts as shown. In this stage, we will put the artificially modified plasmid back into <i>E.coli</i> to express the target product so as to increase the biotin synthase and increase the biotin production. After that, we will use D-caspr 9 technique to block the pathway after the biotin has formed to prevent it from continuing disintegration.
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<h2>Hypothesis for Inhibition of <i>S. aureus</i> by TurboID-AIP Specific Channel Protein Biotinylation</h2>
Here, we invent a new method to block the quorum sensing system in <i>S. aureus</i>. At level 0 which is the rest state, autoinducer protein (AIP) can interact with AgrC, followed by downstream activation of AgrA. AgrA is a quorum-sensing system that levels up the contents of many poisons and virulence determinants when the density of cells raise to a certain amount. AgrA can also amplify the quorum sensing signaling by initiating the expression of downstream gene cluster named agrBCDA. The AgrD protein can interacted with AgrB, then AgrD will be spliced into three parts and AIP can release into the environment to enhance quorum sensing signaling. At level 1, we purified the protein named TurboID-N-AgrD-AIP and these protein can have high affinity on interacting with AgrA through AIP domain and also have high affinity on interacting with AgrD through N-AgrD domain. At level 2, the chemical reaction (other named Protein proximity labeling) happens when we apply the system with biotin and ATP. TurboID will catalyze the biotin and ATP with the lysine residue on the surface of AgrA and AgrB. Those channel proteins have a lot of lysine exposed and will be prevented by interaction with AIP. As such, we can use this specific way to block the the quorum sensing system in <i>S. aureus</i>.
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At level 3, we also hypothesis that if we can link the drug with the streptavidin. Compare with the traditional drug functioning in the body, the interaction between streptavidin with biotinylated AgrA and AgrB. We help the drug target the <i>S. aureus</i> and kill the bacteria at the same time.
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Other application has been performed when we collaborate with Jilin_China team (Level 4). The invent a protein which can eliminate the Pb<sup>2+</sup> in the environment. However, they can not qualify how many bacteria put into the environment can be enough to clean out Pb<sup>2+</sup>. How to qualify the interaction of their LPP-OmpA-pbrR bacteria with Pb<sup>2+</sup>. During the discussion with them, they are quite interesting with our design, so we invent a TurboID-pbrR which is a tool to evaluate how many empty LPP-OmpA-pbrR bacteria. The TurboID-pbrR will help to label the bacteria which have not been absorb with Pb<sup>2+</sup>. In this way, we help them qualify their engineering success at the molecular level.
