Engineering Success | BNUZH-China

Engineering
Degradation Module

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cycle!

Design

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Test

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Cycle 1:Predicting PEBP Sequence

Cycle 2:Modifying the Promoter

Cycle 3:Expressing PE Binding Peptide-PEase Fusion Protein

Cycle 4:Improving the Downstream Assimilation and Mineralization pathways

References

The whole process of biodegradation can be classified into four steps: Biodeterioration, Degradation, Assimilation and Mineralization. Degradation ability of engineered Pseudomonas aeruginosa PAO1 will be gradually strengthened according to the above four steps.

Cycle 1: Predicting PEBP Sequence

Design

According to the needs of the experiment, we need to construct a predictor to determine whether a peptide sequence can bind to PE. After the preliminary literature search, there are 174 pieces of related data collected. Due to the small amount of data, we first performed machine learning training; then we performed special processing and tried to introduce neural networks.

Build

Firstly, the original data is used to train the SVM model; then, the data is increased by 1:10 to get nearly two thousand pieces of data. Then, the trained SVM is used to sift the augmented data according to specific criteria to get a new data set; to dig deeper into the sequence information, the sequence is transformed into a structural graph, the features are extracted using graph neural network (GNN) and location specific scoring matrix, and the attention module is introduced to perform further learning to get a predictor.

Test

The above dataset is divided into training and test sets, experiments are conducted using the built model and corresponding experimental metrics such as accuracy, recall and F1 score are measured.

Learn

Finally, the test results are further analysed and the model parameters are adjusted and optimised.

Cycle 2: Modifying the Promoter

Design

The initial step of constructing a plasmid is finding an appropriate plasmid backbone. We chose pAB1, a broad host range plasmid for IPTG-inducible expression of EGFP, as our basic plasmid backbone for expressing protein in P. aeruginosa. After ordering the plasmid, we found that RBS sequence will be cut off if changes the GFP sequencing to our gene of interest. And if we want to release our engineered bacteria to the soil, a constitutive expression promoter is needed. Replacing the promoter is an urgent need.

Build

We selected 4 promoters that were indicated in the paper that could be expressed in P. aeruginosa, pRPO, Pbla, PoprL and pS.Then linked the promoter sequence into the sequence by enzyme cleavage of NsiI and XbaI on pAB1.

Figure 1 Construction of pAB1-pS plasmid

Test

The pAB1 plasmids with new promoter were transferred into the DH5α strain, and EGFP was successfully expressed.

Figure 2 The DH5α strain with pAB1-pS-egfp and pAB1-P_oprL-egfp plasmids can exsert fluorescence

Learn

When synthesizing short promoter fragments, we used the annealing extension method of two complementary single strand DNA for rapid synthesis. We selected the cleavage sites of NsiI and XbaI for enzyme digestion and ligation operation, replacing the lac operator and ptrc by our aiming promoters. Initially, we tried to use pRPO as a constitutive promoter, but the pRPO promoter could drive the expression of gfp gene in E.coli DH5α strain, but could not drive the expression of the gene in P. aeruginosa PAO1. Therefore, we later found pS, PoprL and Pbla promoters for testing, and found that gfp driven by all three of these promoters could be successfully expressed in PAO1 strain. Given that the pS promoter strain expressed the strongest and most stable fluorescence intensity, we ended up using the pS promoter as our constitutive promoter.

Cycle 3: Expressing PE Binding Peptide-PEase Fusion Protein

Design

After the microorganisms approaching to the microplastics, they can begin the biodeterioration step. Therefore, we intend to construct a fusion protein of PEBP and PE degrading enzymes to enhance the biodeterioration ability of our engineered bacteria. Different kinds of PE degrading enszymes play a role in different situations, so it is crucial to select a suitable PE degrading enzyme, and PEase from wax worm saliva is a suitable candidate. We couple PEBP to PEase. Signal6.0 was used to predict a signal peptide of Esterase-A from P. aeruginosa and bind to the N-terminal of PEBP. The membrane binding domain is predicted using software and is attached to the C end of the PEase. In this way, we have designed a whole PE binding and deteriorate module.

Figure 3 Gene line diagram of pAB1-PEBP-PEase

Figure 4 Gene line diagram of pAB1-PEBP-GFP

Build

We ordered the complete PE adsorption-depolymerization module sequence from a company and cloned it into the pAB1 vector using restriction enzymes. Colony PCR and sequencing confirmed the successful construction of our vector. We introduced an enzyme cutting site between PEBP and PEase to facilitate the replacement of different PEBP sequences. Additionally, we had the company synthesize a sequence with PEBP fused to EGFP to assess the ability of PEBP-expressing bacteria to bind to microplastics.

Figure 5 Construction of pAB1-PEBP-PEase plasmid

Figure 6 Construction of pAB1-PEBP-GFP plasmid

Test

SDS page was used for gene expression detection, and the correct bands of pAB1-PEBP-GFP were obtained, indicating that the protein was indeed correctly expressed in the bacteria. However, we didn't obtain correct band of pAB1-PEBP-PEase plasmid, so for this plasmid we tested it by modeling method.

The PEBP-GFP protein was purified and extracted. From the experimental results, we can clearly see that the fluorescence intensity of PEBP-GFP co-incubated microplastics is obviously higher than GFP co-incubated microplastics and blank control group, indicating that PEBP does play a role in adsorption.

For PEase, we tested its effect through moduling. We conducted structure predicting of PEBP-PEase. The predicting module clearly displayed a membrane spanning domain and an enzyme domain, which we exactly expected our bacteria to produce. After that molecular docking was conducted to study the interactions between the PEase enzyme and both large and small alkane molecules. The result indicating that PEase is able to deteriorate PE plastic.

Learn

In this cycle we successfully constructed pAB1-PEBP-PEase and pAB1-PEBP-GFP plasmids, the effects of PEBP were verified by co-incubation, and PEase was verified by modeling methods.

Cycle 4: Improving the Downstream Assimilation and Mineralization Pathways.

Design

Microplastics are depolymerized into smaller plastic molecules, which then need to be assimilated by microorganisms and further depolymerized within the cells. In the assimilation step, short-chain alkanes can pass through the cell membrane on their own, while medium-short-chain alkanes can enter the cell via AlkB2. For the subsequent mineralization step, we introduced a CYP enzyme with the CYPY96F mutation to efficiently hydroxylate free short-chain alkanes. Medium-long-chain alkanes can be hydroxylated during their entry through AlkB2. To improve the hydroxylation efficiency of medium-long-chain alkanes, we coupled the Rd45 auxiliary factor to AlkB2 to assist with electron transfer. Given that ethanol dehydrogenation is a crucial step in alkane chain mineralization, we also coupled AdhA ethanol dehydrogenase, which can dehydrogenate long-chain alcohols, to AlkB2 to facilitate downstream reactions.

Figure 7 Gene line diagram of pAB1-alkB₂-Rd45-adhA plasmid

Figure 8 Gene line diagram of pAB1-cypY96F-vgb plasmid

Build

We had the company synthesize fragments of the AlkB2-Rd45-AdhA fusion protein and the CYP-VHb fragment, adding enzyme cutting sites at both ends. We ultimately constructed the entire vector using restriction cloning. We obtained the correct colony PCR results and sequencing results, indicating that we successfully constructed the vector.

Figure 9 Construction of pAB1-alkB₂-Rd45-adhA plasmid

Figure 10 Construction of pAB1-cypY96F-vgb plasmid

Test

SDS page was used for gene expression detection, and the correct bands were obtained, indicating that the protein was indeed correctly expressed in the bacteria.

Figure 11(left) Electropherogram of AlkB2-Rd45-AdhA colony PCR gel
Figure 12(right) Electropherogram of CYP96F-VHb colony PCR gel

For Degradation module, we tested its effect through co-incubation. We added 0.1g microplastics into 45ml IS medium, and added 1ml bacterial solution with OD600 of 0.65 for co-culture. After co-incubation for 10 days, Fourier Transform Infrared Spectrometer was used to detect microplastics. It can be found that the peak of carbon-carbon chain in the microplastics of the experimental group gradually decreased with the increase of days, and the oxygen-containing groups gradually increased, and the change was more obvious than that of the control group, indicating that Degradation module can play the role of biodeterioration.

Learn

In this module, we initially wanted to construct a whole plasmid capable of expressing both AlkB2-Rd45-AdhA and CYPY96F as well as VHb proteins. To construct the we needed to attach two long fragments to a vector. At first, we used the enzyme binding method to make the connection, but we tried for a long time without success. It was realized that the method of binding two long f ragments by enzyme digestion was inefficient, and it was difficult to make the vector express three proteins at the same time. We changed the way we connected to homologous recombination and connected one fragment at a time, and we got the engineering success. The effects of AlkB2-Rd45-AdhA and CYPY96F were verified respectively.

References

1. Teufel, F. et al. SignalP 6.0 predicts all five types of signal peptides using protein language models. Nat Biotechnol 40, 1023-1025 (2022).
2. Bergman, M.T., Xiao, X. & Hall, C.K. In Silico Design and Analysis of Plastic-Binding Peptides. J Phys Chem B 127, 8370-8381 (2023).
3. Nie, Y., Liang, J., Fang, H., Tang, Y.Q. & Wu, X.L. Two novel alkane hydroxylase-rubredoxin fusion genes isolated from a Dietzia bacterium and the functions of fused rubredoxin domains in long-chain n-alkane degradation. Appl Environ Microbiol 77, 7279-7288 (2011).

Engineering
Extracellular Electron And CO₂ Transfer

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Cycle 1:Electron Extracellular Transport

Cycle 2:Carbon Dioxide Transfer

References

Cycle 1: Electron Extracellular Transport

In the anoxic mangrove soil environment, Rhodopseudomonas palustris CGA009 requires supplementary electron donors for carbon sequestration alongside carbon dioxide. Consequently, we designed P. aeruginosa PAO1 to act as an electron donor, facilitating electron transfer to R. palustris.

Design

To speed up the carbon sequestration rate of R. palustris, we needed to design a mechanism to allow electrons from copper-green pseudomonas bacteria to flow to R. palustris. After a basic literature search, we learned that direct electron transport and indirect electron transport occurred simultaneously in coculture of P. aeruginosa and R. palustris, coupled with each other to jointly increase the rate of electron transport. Direct electron transfer is carried out by redox proteins (cytochrome c, biological nanowires). Indirect electron transfer is through endogenous electron shuttles (endogenous electron shelters mediated by a benzoyl substance outside the copper-green P. aeruginosa, and photochrome-mediated endogenous electronic shelterings outside the R.palustris bacteria), exogenous electrons shuttle and electron carriers. Therefore, we planned to strengthen both direct and indirect electron transport between P. aeruginosa and R.palustris.

Figure 1 Gene line diagram of pAB1-pS-nqrf plasmid

Figure 2 Gene line diagram of pAB1-pS-pilA plasmid

Build

The introduction of the nqrf gene into P. aeruginosa induces the production of NADH reductase, which is also called NADH dehydrogenase, which catalyzes the transformation of NADH into and electrons, increasing the amount of electrons in P. aeruginosa and thus promoting electron shuttles (PYO), which gene, was introduced into P. aeruginosa, which can express type 4 whiplash on the surface of P. aeruginosa, form physical contact with R. palustris, conduct direct electron transfer, and improve the efficiency of electron transfer between P. aeruginosa and R. palustris.

Figure 3 Plasmid construction of pAB-pS-nqrf

Figure 4 Plasmid construction of pAB-pS-pilA

Test

We used the single fragment homologous recombination method to assemble the plasmid and obtained the correct colony PCR map after many attempts.

Figure 5(left) Electropherogram of pAB1-pS-nqrf colony PCR gel
Figure 6(right) Electropherogram of pAB1-pS-pilA colony PCR gel

Learn

We can find that the nqrf gene can increase the content of NADH reductase in P. aeruginosa and accelerate NADH dehydrogenation to produce and electron content, thereby increasing the advantage of P. aeruginosa as an electrical organism. After the introduction of the type IV whip gene , the surface of P. aeruginosa can express type IV whiplash, form physical contact with R. palustris, conduct direct electron transfer, and improve the efficiency of electron transfer between P. aeruginosa and R. palustris.

Cycle 2: Carbon Dioxide Transfer

Design

At room temperature, carbon dioxide exhibits limited solubility in water, with a ratio of approximately 600:1, primarily forming carbonic acid and trace amounts of carbonates and bicarbonates. However, it readily escapes from aqueous solutions when subjected to heat or agitation, which implies that CO₂ emitted by bacteria engineered for mangrove ecosystems can easily be released into the atmosphere. Furthermore, the degradation process involving P. aeruginosa and R. palustris leads to biofilm formation on microplastics, significantly impeding CO₂ transfer. Consequently, it is essential to convert CO₂ into a form that facilitates efficient transport within these biofilms.

Build

Paeruginosa PAO1 encodes three cytoplasmic β-carbonic anhydrases (β-CAs):psCA1, psCA2, and psCA3, which facilitate the reversible hydration of to bicarbonate, as represented by the equation: $\small{CO_2+H_2O \rightleftharpoons HCO_3^{-} + H^+}$.Notably,psCA1 encoded by the gene PAO102 or psCA1, is a type I β-CA with superior conversion efficiency and pH-independent secondary structure. We utilize psCA1 to convert , generated by P. aeruginosa PAO1 during polyethylene (PE) degradation, into bicarbonate (HCO₃^-), which is then exported via the BicA transporter. R. palustris, a Gram-negative bacterium with cytoplasmic and periplasmic compartments, contains an α-CA (encoded by acaP) in its periplasm. This α-CA is inactive under aerobic conditions but active under anoxic conditions, typical of the low-oxygen environments where mangrove microplastics reside. In R. palustris, α-CA accelerates the conversion of HCO₃^- to , which is subsequently fixed by ribulose-bisphosphate carboxylase-oxygenase (RubisCo) into utilizable organic matter. The ABC transporter substrate-binding protein, a bicarbonate transporter, is present in the cell membrane of R.palustris, enabling the uptake of HCO₃^- and its conversion back to for the CBB cycle by α-CA. To maximize processing from microplastic decomposition, we aim to enhance the HCO₃^-/CO₂.

Figure 7 Plasmid construction of pAB-pS-PAO102

Figure 8 Plasmid construction of pBBR1MCS2-acap

Test

We used both single-fragment homologous recombination and enzymatic ligation methods to assemble the plasmid, and after several attempts, we obtained the correct colony PCR profiles.

Figure 9(left) Electropherogram of pBBR1MCS2-acaP colony PCR gel
Figure 10(right) Electropherogram of pAB1-pS-PAO102 colony PCR gel

Learn

Our molecular experiments have been successfully concluded, yet the forthcoming CO₂ absorption studies are anticipated to demand additional time. As a result, we have not yet been able to allocate resources to functional validation in this domain. However, this represents a significant area of focus for our future enhancements and research endeavors.

References

1. Lotlikar, S.R. et al. Three functional β-carbonic anhydrases in Pseudomonas aeruginosa PAO1: role in survival in ambient air. Microbiology (Reading) 159, 1748-1759 (2013).
2. Lotlikar, S.R. et al. Pseudomonas aeruginosa β-carbonic anhydrase, psCA1, is required for calcium deposition and contributes to virulence. Cell Calcium 84, 102080 (2019).
3. Gorby, Y.A. et al. Electrically conductive bacterial nanowires produced by Shewanella oneidensis strain MR-1 and other microorganisms. Proc Natl Acad Sci U S A 103, 11358-11363 (2006).
4. Pham, T.H. et al. Metabolites produced by Pseudomonas sp. enable a Gram-positive bacterium to achieve extracellular electron transfer. Appl Microbiol Biotechnol 77, 1119-1129 (2008).
5. Ciemniecki, J.A. & Newman, D.K. NADH dehydrogenases are the predominant phenazine reductases in the electron transport chain of Pseudomonas aeruginosa. Mol Microbiol 119, 560-573 (2023).

Engineering
Carbon Fixation

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Cycle 1:Cellulose Production

Cycle 2:NADPH Biosynthesis

References

Cycle 1: Cellulose Production

Design

Within the framework of microbial carbon sequestration, the carbon is predominantly sequestered through the formation of recalcitrant compounds within biological debris. Consequently, augmenting the biosynthesis and aggregation of cellulose, a resilient biopolymer, serves to substantially enhance carbon retention in the soil following the demise of the microorganisms.

Figure 1 Gene line diagram of pBBR1MCS2-bcsA-bcsB plasmid

Build

Cellulose synthesis usually involves multiple subunits, of which BcsA and BcsB are the core catalytic subunits responsible for the synthesis of cellulose chains. Therefore, we increased cellulose synthesis by transferring to bcsA and bcsB derived from Pseudomonas pactida NBRC 14164.

Figure 2 Plasmid construction of pBBR1MCS2-bcsA-bcsB

Test

We employed homologous recombination to successfully ligate the bcsA and bcsB gene fragments into the vector. Subsequent verification through colony PCR and sequencing confirmed the successful ligation of the vector.

Figure 3 Electropherogram of pBBR1MCS2-bcsA-bcsB colony PCR gel

Learn

We initially attempted vector construction through digestion and ligation, but encountered inefficiencies due to the need for promoter replacement. Consequently, we redesigned several primer pairs to facilitate homologous recombination. Despite the vector-to-fragment ratio being nearly 1:1, which resulted in a modest recombination efficiency, our persistent refinement of optimal ligation conditions ultimately enabled us to successfully construct the desired plasmid.

Cycle 2: NADPH Biosynthesis

Design

The efficiency of cellulose production in R. palustris is limited by the intracellular level of NADPH. However, NADPH production pathways are limited. It means that cellulose production is not efficient enough. Therefore, we planned to increase NADPH biosynthesis in R. palustris. Through a literature search, we chose to introduce the nadK gene from R. palustris RCB100, the pntAB gene from Escherichia coli, and the nadM gene from Francisella tularensis into R. palustris. By increasing these three genes, NADPH biosynthesis in engineered bacteria can be enhanced.

Figure 4 Gene line diagram of pBBR1MCS2-nadK-nadM-pntA-pntB plasmid

Build

In R. palustris, Nicotinamide-nucleotide adenylyltransferase (NadM), encoded by the NadM gene, catalyzes the conversion of NMN to NAD⁺ to prevent the depletion of pool. NadK encoded by nadK phosphorylates NAD⁺ to NADP⁺ and pntA-pntB encoded by PntAB further converts NADP⁺ to NADPH. It would increase intracellular levels of NADPH in R. palustris. Increased intracellular levels of NADPH ultimately favor cellulose production.

Figure 5 Plasmid construction of pBBR1MCS2-nadK-nadM-pntA-pntB plasmid

Test

In order to verify the changes in the intracellular level of NADPH, we divided the R. palustris transfected with the target gene into the experimental group and the ordinary R. palustris into the control group. We used the NADP⁺/NADPH detection kit (NADP⁺/NADPH Assay Kit with WST-8) to treated samples and determined changes in the efficiency of NADPH production by comparing the absorbance of the treated bacterial samples. Moreover, we also detected the effects of changes in the intracellular level of NADPH on cellulose production.

Learn

With these experiments, the data we observed indicates a significant increase in intracellular levels of NADPH in the engineered bacteria. It confirms the effectiveness of nadK, nadM, and PntAB.
Due to the low success rate of multi-fragment homologous recombination, we opted to sequentially ligate the inserts, first combining them and then attaching them to the linearized vector. We have effectively concatenated the inserts. However, given the imminent deadline, we faced a time constraint that prevented the completion of the full experiment. We plan to proceed with the remaining experimental work following the iGEM competition.

References

1. Römling, U. & Galperin, M.Y. Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions. Trends in Microbiology 23, 545-557 (2015).
2. Kawai, S., Mori, S., Mukai, T., Hashimoto, W. & Murata, K. Molecular characterization of Escherichia coli NAD kinase. Eur J Biochem 268, 4359-4365 (2001).

Engineering
Safety

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Cycle 1:Primary Designs of the Citrate-activated System

Cycle 2:Optimization of the Promoter of Citrate

Cycle 3:Perfecting of the Suicide System

References

Cycle 1: Primary Design of Citrate-activated System

In order to avoid gene pollution and biological invasion, we design a safety module to ensure engineered bacteria not to make environmental pollution.

Design

In order to settle the problem of gene outflow, we conduct some literature searching and find out several genes for bacteria apoptosis. At first, we select bax as the suicide gene but the engineered bacteria may be extremely burdened by its large size. Therefore, we choose out FADD-DED, a gene that is much smaller than bax but has the similar effect with it.

We still take advantage of P_citH to sense the concentration of citrate and regulate genes downstream. Moreover, we add some genes like T₇ RNA polymerase and cI to construct a plasmid anti-loss system, further preventing gene pollution.

Figure 1 The suicide system under the normal condition

Figure 2 The reaction of the suicide system when lacking in citrate

Figure 3 The plasmid anti-loss function of the suicide system (each cell in the figure beyond represents a situation of plasmid loss)

Build

We divide the gene we interest into two plasmids to lessen the burden on engineered bacteria.

Viewed as a whole, the promoter P_citH is used to start the expression of T₇ RNA polymerase when there is citrate existing in peripheral environment, and then T₇ RNA polymerase will stimulate the promoter pT₇ so that cI can express. The gene cI can produce a kind of substance that specifically repress the activity of P_lam so that it can inhibit the expression of FADD-DED and the bacteria will survive when citrate is in the environment. If engineered bacteria are off the citrate system, the decrease of citrate concentration is not enough to activate P_citH and the downstream expressions stop. Low expression of cI lead to the re-activation of P_lam and FADD-DED is able to come into effect, resulting in cell apoptosis.

Test

To confirm the effect of citrate concentration on P_citH, we designed a series of citrate concentration gradient experiments. We link P_citH with GFP and test the activation of the promoter in different concentration of citrate through the strength of fluorescent light. However, things don't turn out as planned due to insensitivity of P_citH to citrate in P. aeruginosa. We speculate that it may cause by the excessively long sequence we intercept from the upstream of citH and the promoter cannot come into effect therefore.

Learn

We plan to find out other citrate-sensitive promoters through literature searching because of the undesirable sensitivity test of P_citH in order to realize the regulatory of citrate concentration to the suicide system.

Cycle 2: Optimisation of the Promoter of Citrate

Due to the weak sensitivity of P_citHto citrate in P. aeruginosa, we have to replace it by another promoter with similar functions but higher efficiency.

Design

After a long-time literature searching, we select a strong promoter P_opdH from opdH-tctCBA-tctDE manipulator system, which has the similar ability of citrate-sensitivity with P_citH but is smaller in volume and more effective, to replace P_citH. P_opdH natively exists in P. aeruginosa, making sure that it can be activated normally by citrate in the engineered bacteria. Besides, a new type of bacteria (R. palustris) is added to our design and the opdH-tctCBA-tctDE manipulator system is absent in it, which means that we need to introduce the promoter-regulatory gene tctD into R. palustris additionally.

Build

Figure 4 The new regulatory system in P. aeruginosa in normal conditions

Figure 5 The new regulatory system in R. palustris in normal conditions

We follow the previous design of the suicide system but replace P_citH with P_opdH. It is worth mentioning is that we link the tctD upstream of P_opdH in R. palustris so that the promoter is able to be regulated more accurately.

Figure 6 The mechanism of the new system when there is no citrate (in P. aeruginosa)

Figure 7 The mechanism of the new system when there is no citrate (in R. palustris)

Test

To confirm the effect of citrate concentration on P_opdH, we designed a series of citrate concentration gradient experiments. We link P_opdH with GFP and test the activation of P_opdH in different concentration of citrate through the strength of fluorescent light. Unfortunately, there is a critical deficiency in our design that FADD-DED has a long sequence of C and G base pairs, which makes it hard to synthase by the method of PCR. Besides, our design of the plasmid anti-loss system is too large to realize in labs. For these reasons we have to adjust some genes we apply and haven't design corresponding test of FADD-DED in the direction of wet lab and dry lab.

Learn

Over these experiments, we learn the importance of simplifying the system, and we try to design a more straightforward but still efficient safety module.

Cycle 3: Perfecting of the Suicide System

Owing to the unrealizability of the previous design in labs as well as the adaptability of the system in engineered R. palustris, we urgently need an easier and more generic method to realize our wish.

Design

We introduce hok/sok toxin-antitoxin system to achieve both of plasmid anti-loss and bacterial suicide when being out of the mangrove at the same time. The expression (sok antisense RNA and hok mRNA) will specifically bind to each other and the production of hok toxin is inhibited. However, the half-life of sok antisense RNA is much shorter than that of hok mRNA, which results to the earlier degradation of sok antisense RNA than hok mRNA when the plasmid is lost. The hok mRNA remaining is able to encode hok toxin and lead to cell death. We replace the promoter of sok with P_opdH and the promoter of hok is replaced by a weak promoter PcW common in P.aeruginosa as well, ensuring the expression strength of sok to be always stronger than the one of hok in normal conditions and cells keeping living. When engineered bacteria is out of the mangrove, the decrease of citrate concentration will affect the expression of sok, causing a lower expression level of sok antisense RNA. Some of the hok mRNA is released thus and generates toxin to kill the bacteria.

Build

To secure the plasmid anti-loss effect of the hok/sok system, we attach them to the same plasmid.

Figure 8 The new suicide system under normal conditions

Figure 9 The effect of the new suicide system when the plasmid is lost

Figure 10 The new suicide system when lacking in citrate

Test

To test the plasmid anti-loss effect of hok/sok system, we design a series of experiments. First, we introduce the hok/sok system into the experimental group while the empty plasmid without the system is introduced into the control group, which enable us to view the killing effect of our design. We take advantage of the LB liquid culture media to cultivate the both groups, deliberately making the plasmids in bacteria lost. Passing the bacteria on until they reach the tenth generation, we transfer them on the LB solid culture media, cultivating in 37℃ for 16h. Then move the single colonies to those with ampicillin, cultivating in 37℃ for 6h. Finally, we count the number of colonies on the plate with ampicillin, and separately calculate the mortality rate of two groups.

From those test plates, we can make a conclusion that the mortality rate of the group without hok/sok system is larger than the one with the system. It means that the number of plasmid-loss bacteria without the system is more than the experimental group. Therefore, the function of preventing plasmid loss is effective.

Then we conduct experiments to test the suicide function when the engineered bacteria are out of the mangrove.

Figure 11 (A) The death rate of the groups with and without hok/sok system, which also means the rate of plasmid loss. (B) OD600 of the groups with and without IPTG induction, which is designed as a substitute experiment in the laboratory since citrate can be consumed by P. aeruginosa

Learn

We successfully construct the safety module.

References

1. Thisted, T. & Gerdes, K. Mechanism of post-segregational killing by the hok/sok system of plasmid R1. Sok antisense RNA regulates hok gene expression indirectly through the overlapping mok gene. J Mol Biol 223, 41-54 (1992).
2. Franch, T., Gultyaev, A.P. & Gerdes, K. Programmed cell death by hok/sok of plasmid R1: processing at the hok mRNA 3'-end triggers structural rearrangements that allow translation and antisense RNA binding. J Mol Biol 273, 38-51 (1997).
3. Brocker, M., Schaffer, S., Mack, C. & Bott, M. Citrate utilization by Corynebacterium glutamicum is controlled by the CitAB two-component system through positive regulation of the citrate transport genes citH and tctCBA. J Bacteriol 191, 3869-3880 (2009).