Electron transfer system

The key point of achieving the goal of reductive degradation of azo dyes is to gain electrons, and here comes our chassis organism, the DMRB bacterium Shewanella oneidensis MR-1, which can perform anaerobic respiration by utilizing some metal ions or minerals such as Fe(III) as terminal electron acceptors in the absence of other electron acceptors. S. oneidensis MR-1 can transfer electrons through Mtr-pathway, endowing itself great capability of reduction^[1]. Therefore, we planned to select azo dye as the electron acceptor instead of metal minerals so that the azo bond can be reduced and broken. In order to enhance the reduction ability of S. oneidensis MR-1 and make it more suitable for application, we designed an automatic control system, through harnessing the quorum-sensing (QS) system to tune the Mtr electron conduit pathway. The details of the system are described as follows.

Figure 1. Mechanism of Mtr

The components of the Mtr path-way have been identified to include CymA, MtrA, MtrB, MtrC, and OmcA. CymA is an inner-membrane tetraheme c-type cytochrome (c-Cyt) that belongs to the NapC/NrfH family of quinol dehydrogenases. It is proposed that CymA oxidizes the quinol in the inner-membrane and transfers the released electrons to MtrA either directly or indirectly through other periplasmic proteins^[2]. A decaheme c-Cyt, MtrA is thought to be embedded in the trans outer-membrane and porin-like protein MtrB. Together, MtrAB deliver the electrons through the outer-membrane to the MtrC and OmcA on the outmost bacterial surface. Functioning as terminal reductases, MtrC and OmcA can transfer electrons directly to azo dye via their solvent-exposed hemes because of their extracellular location and broad redox potentials.
Considering CymA will oxidize quinol in the inner-membrane^[2] and so its overexpression may pose great threat to the redox balance in our chassis, we chose not to overexpress this inner-membrane protein and only to overexpress the other four outer-membrane proteins: MtrCAB complex and OmcA. Then, MtrCAB can be isolated as a protein complex with a ratio of 1:1:1^[3] and it is proposed that MtrB is a trans outer-membrane spanning β-barrel protein that serves as a sheath to embed MtrA in the membrane where MtrAB form a trans outer-membrane delivery module for transferring electrons to MtrC, which functions as an extracellular reductase^[4]. Therefore, we’d like to keep the original expression ratio of MtrA/B/C to make more MtrCAB complex, so we keep the original RBS of these 3 genes in the genome of S. oneidensis MR-1 (universally, original RBS is within 30bp before CDs). As for OmcA, though it can form a stable complex with MtrC with the ratio of 2:1^[5], no direct evidence shows that it’s necessary for its function as a terminal reductase. Thus, we just equipped it with a strong RBS B0034. Here is our overexpression DNA design.

Figure 2. Gene Circuit of Mtr

Our adviser Yu suggested us that the overexpression of the outer-membrane protein, OmcA in particular, will induce great stress to bacteria, even lethal (has been comfirmed previously by experiment). In order to decline the metabolic stress of our chassis host during growth, we harness luxR-luxI quorum-sensing system to control the overexpression of the Mtr pathway, so that the strengthened overexpression of Mtr pathway will only be initiated until bacterial growth reached a particular level. We integrated the luxI/luxR genes into the genome of S. oneidensis MR-1 and use pluxI promoter to promote our overexpression ORF.

Figure 3. QS System

Aromatic amines resistance system(aNAT)

The toxicity of aromatic amine on bacteria

After the azo dyes have been broken up, two molecules of aromatic amines generated. As this chemical compound is structurally similar to aminobenzoic acid (PABA), it can have competitive effect on bacterial PABA dihydrofolate synthetase, preventing PABA folic acid as raw materials for the bacteria to produce folic acid. It can also reduce the amount of metabolically active tetrahydrofolic acid, which is the significant raw material for the synthesis of purines, thymine nucleoside and DNA for bacteria. Therefore, the growth of bacteria is inhibited. Though aromatic amines are easily oxidized by oxygen in the air, it is difficult to be degraded by Shewanella oneidensis MR-1 under an anaerobic environment. What`s worse, the accumulation of aromatic amine will limit the growth of the bacteria, which can lower the efficiency of azo dye reduction.

Convenient methods of bacterial degradation

Current methods for the bacterial degradation of aromatic amine focus on using several bacteria with unique pathways which can degrade aromatic amines like aniline^[5]. But nearly all of the degradation pathways need an aerobic environment and are complicated. Therefore, it is difficult for the pathways to be transformed and fully functional under anaerobic environment.

The aromatic amine N-acetyl transferase (aNAT)

To solve this problem, we use the aromatic amine N-acetyl transferase (aNAT) to "destroy" the aromatic amines. The aNATs can transfer an acetyl group from substrates like Acetyl-CoA to the amidogen to form an acylamino, thus the "active" hydrogen atom of the amidogen is substituted. Therefore the amidogen cannot damage the growth of S. oneidensis MR-1.

Figure 4. Aromatic amines resistance system(aNAT)

Figure 5. Mechanism of aNAT

Glucose Utilization

For many Shewanella spp., their genomes lack the key enzymes in glycolysis, Shewanella spp. strains prefer two- and three-carbon carbohydrates as carbon or electron source. In order to achieve glucose utilization in Shewanella oneidensis MR-1, our team introduce two essential genes——the glucose facilitator (glf) and hexokinase(HXK) to construct the engineered S. oneidensis MR-1.

Gene HXK (standing for Hexokinase) acquiring from our PI catalyzes the phosphorylation of glucose to form Glucose-6-phosphate while consuming a molecule of ATP. The free energy of this reaction is very low, for which it is usually thought as irreversible. It also serves as the first step of glycolysis——once the catalyzation takes place, subsequent steps of glycolysis can proceed rapidly.

Glf from E. coli makes it possible for S. oneidensis MR-1 to transport glucose into cells without consuming ATP.

We planned to insert those two genes into genome to produce a stable new strain, so we constructed a knock-in plasmid. The upstream and downstream sequences are carefully searched from S. oneidensis MR-1 genome so that inserted fragment will be placed at the proper site on the S. oneidensis MR-1 genome.

Figure 6. Glucose Utilization

Light Activated Kill Switch

Our bacteria is designed to be used in anaerobic tower at waste water treatment plant. (Click here to learn about the details) To prevent our bacteria from escaping to environment, we decided to introduce a light activated kill switch. We found one from Team Unesp Brazil 2018.

Mechanism

Photoreceptor VVD is used as the “switch”[2]. The engineered variants are named Magnets (pMag and nMag). Positive (BBa_K2660009) and negative (BBa_K2660008) magnets (pMag and nMag) dimerize in response to blue light through electrostatic interactions (Fig. 1).

Cas9 is used as the “killer”. Guided by gRNA, Cas9 will cut down the target sequences, causing cell death. N-terminal and C-terminal domain of Cas9 are separately linked to magnets by a 10 aa linker (BBa_K105012). Therefore, Cas9 is activated only when pMag and nMag dimerize under blue light.

Design of gRNA

The gRNA consists of target, PAM, and scaffold sequences. To ensure cell death when necessary, we targeted essential genes like RNA polymerase B (rpoB), DNA polymerase III and DNA ligase. We designed the target and PAM sequences with the help of an online tool[3]. Two versions of gRNA were constructed, with the highest or second highest estimated on target rate (Table 1). And the sequence of scaffold was found from a handbook.

Table 1 Estimated on target rate

	Ver 1	Ver 2
rpoB	1	1
DNA polymerase III	0.96	0.94
DNA ligase	0.88	0.88

Figure 7. Sequences of gRNA

Figure 8. Light Activated Kill Switch

Future work

As mentioned earlier, we have designed the “Electron Transfer System” to enhance the ability of Shewanella oneidensis MR-1 to degrade azo dyes. And we have achieved our goal satisfactorily. However, inspired by the result of our modeling, we think that we can make our system work better, which means stronger ability of degrading azo dyes. Therefore, if time permits, we will overexpress CymA in Shewanella oneidensis MR-1. But considering the surviving stress brought by the expression of CymA, we hope to control the ratio of the expression of CymA, MtrA, MtrB, MtrC and OmcA. So we think that we can replace the RBS in front of CymA, MtrA, MtrB, MtrC and OmcA with standard RBS provided by iGEM. We plan to use four RBS, respectively called B0034, B0064, J61101, J61117. As we all know, their abilities of improving the expression of gene are different. Thus, if we add them respectively in front of CymA, MtrA, MtrB, MtrC and omcA, depending on the result of modeling and the expression efficiency of these RBS, we may get a Shewanella oneidensis MR-1 with stronger ability of degrading azo dyes. Besides, considering that we only make the model of CymA, MtrA and MtrB, we want to add different RBS in front of CymA, MtrA, MtrB, MtrC and OAmcA. Because of different expression efficiency of each RBS, different combination will cause different expression intensity of CymA, MtrA, MtrB, MtrC and OmcA. Although we don’t know which kind of ratio in the expression of five gene is the best occasion for the ability of degrading azo dyes, we can gain a Shewanella oneidensis MR-1 stronger ability of degrading azo dyes definitely possibly. After we gain the best result, we can compare it with our modeling result to verify the correctness of our modeling.

Reference

[1] Liang Shi, Kevin M. Rosso, Tomas A. Clarke, David J. Richardson, John M. Zachara and James K. Fredrickson (2012). Molecular underpinnings of Fe(III) oxide reduction by Shewanella oneidensis MR-1. frontiers in Microbiology, published: 15 February 2012. doi: 10.3389/fmicb.2012.00050

[2] Field, S. J., Dobbin, P. S., Cheesman, M. R., Watmough, N. J., Thom-son, A. J., and Richardson, D. J. (2000). Purification and magneto-optical spectroscopic characterization of cytoplasmic membrane and outer membrane multiheme c-type cytochromes from Shewanella frigidimarina NCIMB400. J. Biol. Chem. 275, 8515–8522.

[3] Ross, D. E., Ruebush, S. S., Brantley, S. L., Hartshorne, R. S., Clarke, T. A., Richardson, D. J., and Tien, M. (2007). Characterization of protein-protein interactions involved in iron reduction by Shewanella oneidensis MR-1. Appl. Environ. Microbiol. 73, 5797–5808.

[4] Hartshorne, R. S., Reardon, C. L., Ross, D., Nuester, J., Clarke, T. A., Gates, A. J., Mills, P. C., Fredrickson, J. K., Zachara, J. M., Shi, L., Beliaev, A. S., Marshall, M. J., Tien, M., Brantley, S., Butt, J. N., and Richardson, D. J. (2009). Characterization of an electron conduit between bacteria and the extracellular environment. Proc. Natl. Acad. Sci. U.S.A. 106, 22169–22174.

[5] Cocaign A, Bui LC, Silar P, et al. Biotransformation of Trichoderma spp. and their tolerance to aromatic amines, a major class of pollutants. Appl Environ Microbiol. 2013;79(15):4719–4726. doi:10.1128/AEM.00989-13

[6] https://2018.igem.org/Team:Unesp_Brazil/Design

[7] KAWANO, F. et al. Engineered pairs of distinct photoswitches for optogenetic control of cellular proteins. Nat. Commun. v.6 n.6256, 2015.

[8]https://sg.idtdna.com/site/order/designtool/index/CRISPR_SEQUENCE

Team:USTC/Design