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To construct a selection module, we need to find a gene circuit which can respond to alkanes, our target products. In nature, many oil-degrading prokaryotes such as P. putida Gpo1, Alcanivorax borkumensis SK2 and Acinetobacter baylyi ADP1 have gene circuits responding to alkanes(alkS-alkB from P. putida Gpo1, alkS-alkB1 from Alcanivorax borkumensis SK2[1], and alkR-PalkM from Acinetobacter baylyi ADP1[2]). We choose the alkR-PalkM circuit in Acinetobacter baylyi ADP1 which has remained untouched in iGEM competition, because this circuit is specifically suitable for alkane synthesis pathway. AlkR responds to a broad range of alkanes with carbon chain length from C7 to C36, and it’s the only bioreporter that is able to detect alkane with carbon chain length greater than C18[2]. Additionally, although fatty alcohol whose structure is similar to alkane can also combine with alkR but their combined compounds will inhibit PalkM.
Alkanes are presumably recognized by alkR and their interaction triggers a conformation change of alkR dimers which leads to the activation of genes downstream of promoter alkM[2].
To know more about Acinetobacter baylyi ADP1, alkR protein, and P-alkM promoter, please clike here.
To realize the goal of selection, we construct two circuits based on the interaction mechanism of alkR and PalkM.
The first circuit is a fluorescent selection circuit. We put the RFP(red fluorescent protein) gene under the regulation of PalkM. When PalkM is induced, downstream RFP gene will express, and red fluorescence of cells can be detected. Therefore, we can transform the alkane molecules into fluorescent output.
The second circuit is a resistant selection circuit. We replace TetA gene with RFP(red fluorescent protein) gene. TetA will transport tetracycline out of the cell so that the intracellular concentration of tetracycline is relatively stable, and the growth of cells will not be inhibited. Thus, we can turn alkane molecules into the output of the resistance against tetracycline of cells.
To know more about the transportation mechanism of tetA protein, please click here.
The two selection circuits mentioned above can be applied to irrationally modify alkane producing module.
First method is to combine the strains with an alkane producing module and a fluorescent selection circuit in the medium. Theoretically, the strains with higher alkane yield will have a stronger expression level of RFP. We can select out the high yield strains which have strong fluorescence indensity.
Another method is to put the strains with an alkane producing module and a resistant selection circuit in the medium of a certain concentration of tetracyclin, hoping that strains with improved alkane productivity will have a stronger capacity to suivive and enrich under this kind of selection pressure. After several rounds of enrichment culturing, we can get strains with high alkane productivity.
Introduction of alkR and PalkM
Figure 1. Homologous modeling figure of protein ALKR
Protein ALKR and promoter alkM are originally found from Acinetobacter baylyi ADP1.
Acinetobacter sp. are ubiquitous bacteria in natural aquatic and soil environment that are frequently found to be capable to degrade a broad range of carbon chain alkenes and alkanes[3]. And Acinetobacter baylyi ADP1 is able to use long-chain-length alkanes with at least 12 carbon atoms as the sole source of carbon and energy[4].
In Acinetobacter baylyi ADP1, P-alkM is the promoter of alkane hydroxylase genes which encodes alkane hydroxylase(alkM), a crucial enzyme in the degradation of alkanes. AlkM, together with rubredoxin (RubA) and rubredoxin reductase(RubB) forms a three-component alkane monooxygenase complex which oxidate inert alkane to the respective primary alcohol[4].
AlkR is an AraC/XylS-like transcriptional regulatory protein. It includes C-terminal DNA-binding domain for promoter binding and N-terminal domain for inducer recognition[2]. Its function is to recognize alkane molecule after its own dimerization and induce promoter alkM. The activity of alkane hydroxylase could impede ALKR’s function.
Introduction of TetA
Figure 2. Efflux mechanism of tetracycline resistance
Protein TetA is a tetracycline cation/proton antiporter located in the cell membrane. When there is no tetracycline within the cell, inhibitor protein TetR combine with gene TetO and inhibit the expression of. TetA and TetR. When diffusing into the cell, tetracycline combines with Mg2+ and forms Mg2+-tetracycline complex. The complex combine with TetR and turn on the expression of TetA and TetR. And then tetA pumps out the complex and pumps in H+[5].
With the decreasing of Mg2+-tetracycline complex within the cell, there are more TetR combining with gene tetO and the expression of tetR and tetA is turned off. And the system returns to steady state.
Reference
[1] Rekha Kumari, Robin Tecon, Siham Beggah et al.(2011) “Development of bioreporter assays for the detection of bioavailability of long-chain alkanes based on the marine bacterium Alcanivorax borkumensis strain SK2.” Environmental Microbiology 13(10), 2808–2819
[2] Zhang, Dayi, et al.(2012) "Whole-cell bacterial bioreporter for actively searching and sensing of alkanes and oil spills." Microbial Biotechnology 5.1: 87-97.
[3] Young, David M., Donna Parke, and L. Nicholas Ornston. (2005) "Opportunities for genetic investigation afforded by Acinetobacter baylyi, a nutritionally versatile bacterial species that is highly competent for natural transformation." Annu. Rev. Microbiol. 59: 519-551.
[4] Ratajczak, Andreas, Walter Geißdörfer, and Wolfgang Hillen. (1998) "Expression of Alkane Hydroxylase fromAcinetobacter sp. Strain ADP1 Is Induced by a Broad Range of n-Alkanes and Requires the Transcriptional Activator ALKR."Journal of bacteriology 180.22: 5822-5827.
[5] Speer, Brenda S., Nadja B. Shoemaker, and Abigail A. Salyers. (1992)"Bacterial resistance to tetracycline:mechanisms, transfer, and clinical significance."Clinical microbiology reviews 5.4: 387-399.
