Difference between revisions of "Team:NKU China/Description"

As illustrated in Panel A, there are mainly 2 steps involved in the processing of AI-2 production in bacteria cells. AI-2 is produced from S-adenosylhomocysteine (SAH) by Mtn and LuxS and accumulates extracellularly with cell density. In our project, we cloned mtn, luxS into the plasmid pTrcHisB to enable overexpression of proteins associated with these AI-2 production machanisms in E. coli. Two AI-2 Supplier Device, pLuxS and pLuxSMtn were successfully constructed using homologous recombination technology.

Scheme 1. Panel (A) depicts two AI-2 Supplier Device, pLuxS and pLuxSMtn we constructed in our project. Panel (B) depicts the AI-2 Supplier cells that are engineered through the overexpression of distinct components of AI-2 production pathway. "Native" indicates native production, while "Induced" indicates over-expression. Panel (C) illustrate how AI-2 Supplier devices work in our chassis.
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During the construction, all constructs and final plasmids were immediately verified by a restriction digest and/or sequencing (details can be accessed in our Notebook).

As illustrated in Panel A, there are mainly three steps involved in the processing of AI-2 from the extracellular environment are uptake, primarily through the LsrACDB transporter, (ii) LsrK-mediated phosphorylation of AI-2 (to AI-2P), which blocks export back to the extracellular milieu so that accumulated AI-2P binds the regulatory protein LsrR, derepressing the Lsr transporter as well as enzymes, LsrF and LsrG, and (iii) degradation of AI-2P through the two step process from isomerase LsrG followed with cleaving and thiolation by LsrF. In our project, we cloned lsrACDB, lsrK, lsrFG into the plasmid pTrcHisB to enable overexpression of all proteins associated with these AI-2 processing mechanisms in E. coli. Six AI-2 Supplier Device, pLsrACDB, pLsrK, pLsrFG, pLsrACDBFG, pLsrACDBK, pLsrACDBFGK were successfully constructed using homologous recombination technology.

Scheme 2. Panel (A) depicts six AI-2 Supplier Device, pLsrACDB, pLsrK, pLsrFG, pLsrACDBFG, pLsrACDBK, pLsrACDBFGK we constructed in our project. Panel (B) depicts the AI-2 Consumer cells that are engineered through the overexpression of distinct components of AI-2 production pathway. 'Native' indicates native production, while 'Induced' indicates over-expression. Panel (C) illustrate how AI-2 Consumer devices work in our chassis.
See more in Proof of Concept.

For AI-2 Response Device A, this composite part consists of the AI-2 (autoinducer-2) quorum sensor-inducible promoter BBa_K1981101, a GFP coding sequence BBa_E0040, a double terminator BBa_B0015. In AI-2 Response Device A, GFP expression is under the control of promoter, plsr. When phospho-AI-2 binds LsrR, expression of GFP ensues. The expression of GFP can directly response to the AI-2 level in the environment, which is an alternative way to reflect the AI-2 concentration in the nature or artificial environment.

And in AI-2 Response Device B, additional lsrR expression enables additional repression of target genes for tighter regulation and delayed response compared to AI-2 response device A.

Scheme 3. Panel (A) depicts that plsr region which is depressed by the response regulator LsrR. AI-2P depresses the response regulator LsrR, thereby activating transcription of the lsr operon. Panel (B) depicts the partes that we succeefully cloned for construction of AI-2 Response Devices. Panel (C) illustrate the AI-2 Response devices we constructed on pTrcHisB.
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Pioneering biocontainment systems used metabolic auxotrophy in which target cells could only grow in the presence of an exogenously supplied metabolite, and the recent creation of an E. coli strain with an altered genetic code enabled production of synthetic auxotrophy strains that require an exogenous supply of non-natural amino acids for cell survival.

Traditional metabolic auxotrophy strains are hampered by the potential for inadvertent complementation by cross-feeding or by the presence of the metabolite in heterogenous environments, and synthetic auxotrophy systems rely on extensive genome-wide engineering that may be impractical for many industrial production and biotherapeutic microbes. Furthermore, they are intrinsically difficult to reprogram for different environmental conditions, potentially limiting their application.

An alternative approach to biocontainment is to use gene circuits to maintain essential gene expression or block toxin gene expression under the assigned biocontainment conditions. Upon loss of the biocontainment signal, the circuit blocks essential gene expression or induces toxin gene expression to kill the cell.

Here we present a synthetic biosafety degradation system based on the Gram-positive M. florum tmRNA system that does not rely on host degradation systems and can function in a wide range of bacteria. Researchers showed that the M. florum ssrA tag (mf-ssrA) is degraded by its endogenous Lon protease (mf-Lon) but not by E. coli Lon or ClpXP, and mf-Lon does not recognize or degrade ec-ssrA, providing a protease and cognate degradation tag with orthogonal functionality in E. coli.

In our biosafety system, firstly, we use CRISPR/Cas9 technology to add mf-lon ssrA tag into 5 essential genes. Then by applying gene circuits to control mf-lon protease expression under the assigned biocontainment conditions, we can blocks essential gene expression to kill the cell upon loss of the biocontainment signal.

Scheme 4. Panel (A) depicts that the kill switch which controls the expression of mf-Lon gene. Without IPTG, the promoter Pgrac is inhibited by suppressor LacI and the supreessor XylR will not synthesized, thus the promoter Pxyl is active and mf-Lon gene is expressed. When IPTG is added, the xylR gene is expressed and the suppressor XylR is synthesized thereafter inhibited the expression of mf-Lon genes. Panel (B) depicts tive essential gene was targeted to add the the mf-ssrA tag. Panel (C) illustrates schematic of biosafety system in which IPTG-induced mf-Lon expression allows the mf-Lonprotease to degrade essential proteins in a mf-ssrA-dependent manner. We use CRISPR/Cas9 technology to add mf-lon ssrA tag into 5 essential genes.
See more in ，Proof of Concept.