Introduction

We hope to build a co-culture system of Synechocystis sp. PCC 6803 and S. oneidensis MR-1, which can use solar energy to fix carbon dioxide to generate electricity. Therefore, we designed several components and four system paths to achieve this. In the build phase, we first used homologous recombination to build these pathways but we encountered some problems. Through continuous trials and summaries, we switched to using the double enzyme digestion and enzyme ligation to construct our pathway, and finally achieved certain results. Our experiments would provide some experience for subsequent teams.

CYCLE 1: constructed by homologous recombination method

1.1 Design

To construct a well-functioning system pathway, we hope to utilize four plasmids to improve the efficiency of the electricity production of S. oneidensis MR-1, enhance the intensity of Synechocystis sp. PCC 6803 photosynthesis, and increase the antioxidant activity of Synechocystis sp. PCC 6803 and the stress resistance of S. oneidensis MR-1 respectively. Based on existing parts with independent functions, we achieve our goal by connecting multiple components to complete plasmids to form a fully functional system pathway.

We wanted to build our plasmid by using homologous recombination. Therefore, we introduce homologous arms into the components when building individual elements, which is the basis of the entire pathways construction.

First, In the pLactate pathway, we begin by incorporating the first homologous arm downstream of the slr0168 vector fragment, which includes the ampicillin-resistant gene, and upstream of ldhA-lldP-omcs. Following that, a second homologous arm is introduced downstream of ldhA-lldP-omcs and upstream of the slr0168 vector fragment.

In the pResistance6803 pathway, we add the first homologous arm to the downstream of the slr2030 vector fragment containing the ampicin and kana resistant genes and upstream of dsup-gshA-gshB. A second homology arm is then added downstream of dsup-gshA-gshB and upstream of the slr2030 vector fragment.

In the pResistance MR-1 pathway, we add the first homologous arm to the downstream of the PYYDT vector fragment containing the kana resistance gene and upstream of dsup-sodA-oprf. A second homology arm is then added downstream of dsup-sodA-oprf and upstream of the PYYDT vector fragment.

In the pElectricty pathway, we add the first homologous arm to the downstream of the PYYDT vector fragment containing the kanamycin resistance gene and upstream of ycel-pncB. A second homology arm was added downstream of ycel-pncB and upstream of nadD-nadE-nadM, and a third homology arm was added downstream of nadD-nadE-nadM and upstream of the PYYDT vector fragment. Homologous arms are added to different gene fragments by PCR using different primers. In principle, these specific homologous arms ensure our experimental feasibility.

1.2 Build

Based on the principle of seamless cloning, we tried to use homologous recombinase to attach multiple fragments directly into the vector to form a complete plasmid.

Take the pElectricty system pathway as an example: first, the linearized vector PYYDT and gene fragments - ycel-pncB and nadD-nadE-nadM were obtained by PCR. They were then validated and purified by gel extraction. Finally, homologous recombinase was used to directly connect the linearized vector PYYDT with two fragments of ycel-pncB and nadD-nadE-nadM to construct a complete system pathway. We then transfered the homologous recombinant product into E. coli TOP10 and seed into LB plates containing 50 μg/mL kanamycin for screening. When single colonies appeared on the surface of solid media, we singled out the single colonies for PCR validation. After observing the correct bands, we mixed 800 μl of bacterial solution with 800 μl of 1:1 mixture of glycerol and double distilled water and keep the seeds at -80℃.

1.3 Result

We tried to construct our pathway through multi-fragment homologous recombination. After several attempts, we successfully constructed plasmids omcs, ycel-pncB and nadE-nadD-nadM.

1.4 Experience

After several attempts, we found that the direct use of multi-fragment homologous recombination is not suitable for most pathways, so we carefully analyzed and summarized the reasons for the failures. We believe that the success rate of homologous recombination is low due to the large number of genes, the huge number of bases and the complex topology contained in the pResistance MR-1, pLactate, pResistance 6803, pElectricity pathways. Therefore, we tried to convert to the method of enzyme digestion and ligation to solve this problem.

CYCLE 2: constructed by double enzyme digestion and enzyme ligation method

2.1 Design

After many failed attempts, we decided to try double enzyme digestion and enzyme ligation to construct our plasmids.

We first perform a double enzyme digestion reaction to obtain multiple fragments and vectors with the same sticky ends, and then ligate the carriers and fragments containing the same sticky ends together through an enzyme ligation reaction. We hope it will solve the problem of low success rate of homologous recombination in dealing with multi-fragment and large-fragment at this stage.

In the first step, we use the double enzyme digestion method to obtain the target gene fragments and the vector fragment with sticky ends. We first culture the basal plasmid-containing strain and extract the plasmid, then add the digestion site to the target gene fragment and the vector fragment by PCR, and then obtain the target gene fragment and the vector fragment with the same sticky end by double enzyme digestion. For dsup-gshA-gshB fragments for Synechocystis sp. PCC 6803 and ycel-pncB-nadE- nadD-nadM fragments for S. oneidensis MR-1, we directly cleavage the extracted basal plasmid by double enzyme digestion to obtain gene fragments with sticky ends, For the ldhA-lldP-omcs fragment for Synechocystis sp. PCC 6803 and the dsup-sodA-oprf fragment for S. oneidensis MR-1, we first PCR the extracted plasmid to obtain the target gene fragments, and then obtain the fragment with sticky end by double enzyme digestion.

In the second step, we use enzyme ligation to link the target gene fragment and its corresponding vector fragment with the same sticky end together, and finally construct a complete plasmid.

2.2 Build

Our basic process of constructing a complete plasmid is: extract the plasmid, perform PCR to obtain the fragment containing the target gene, obtain the fragment of the target gene with sticky ends through double enzyme digestion, and use the enzyme ligation reaction to link the gene of interest to the vector.

Take the pLactate system pathway as an example: we first obtain linearized vector fragments containing ampicure resistance by PCR, and then obtain gene fragments and vector fragments by gel extraction, then verify and purify gene fragments and vector fragments. We then double-enzyme-digested the fragments with restriction enzymes SpeI and EcorI to obtain gene and vector fragments with identical sticky ends. Finally, we used an enzyme ligation reaction to ligate a fragment containing the gene ldhA-lldP-omcs with the vector fragment corresponding to it to construct a complete plasmid. We then transferred the constructed plasmid into E. coli TOP10 and seeded into LB plates containing 100 μg/mL ampicin for screening. When single colonies appeared on the surface of solid media, we singled out the single colonies for PCR validation. After observing the correct bands, we mixed 800 μl of bacterial solution with 800 μl of 1:1 mixture of glycerol and double distilled water and keep the seeds at -80℃.

2.3 Result

After several attempts, we successfully constructed plasmids pLactate, pResistance6803, pElectricity, pResistance MR-1.

2.4 Experience

At first, we encountered the problem that the digestion reaction could not be carried out due to the low concentration of the recovered PCR fragments. After several attempts, we found that the extraction of PCR fragment products, the timing and temperature of the double enzyme digestion reaction and the enzyme ligation reaction had a great influence on the success rate of plasmid construction. Following numerous setbacks, we meticulously analyzed and documented the underlying causes. After conducting a series of experiments, we successfully extracted the necessary fragments and vectors from fluid samples, maximizing their concentrations for double enzyme digestion. After double enzyme digestion, we obtained the double enzyme digestion product by gel extraction to ensure the purity of the fragment. At the same time, we also continuously adjusted the reaction temperature and narrowed The time range of the reaction, and finally found the most suitable reaction time and temperature system. After a series of difficulties, we figured out an optimally constructed system and obtained the corresponding complete plasmid.

Electrico sisters is of great significance to solve the energy crisis and protect the global environment, encouraging us to use synthetic biology to make some contributions in this regard. We will continue to carry out this topic and are willing to do our part for the development of synthetic biology.

CYCLE 3: Expression validation

3.1 Synechocystis sp. PCC 6803

3.1.1 Design

We hope to figure out whether the growth cycle of Synechocystis sp. PCC 6803 engineered bacteria is shorter than that of wild-type Synechocystis sp. PCC 6803. Therefore, we detect the growth of Synechocystis sp. PCC 6803 and plot its growth curve. At the same time, we verify whether the efficiency of the strain we constructed achieves our expectations by detecting the concentration of expression products.

3.1.2 Build

First, we culture the E. coli TOP10 strain containing the correct plasmid and extracted the plasmid. Next, we transfer the extract plasmid into Synechocystis sp. PCC 6803. The experimental operation is: we use BG11 liquid medium to resuspend and wash wild-type Synechocystis sp. PCC 6803 twice, take an appropriate amount of Synechocystis sp. PCC 6803 solution and add an appropriate amount of plasmid, stand and activate it for a period of time, apply the bacterial solution to BG11 plate with corresponding resistant. When the transformant appears on the surface of the solid medium, we pick out the transformant for PCR verification. After observing the correct bands, we mix 800 μl of Synechocystis sp. PCC 6803 solution with 800 μl of 1:1 mixture of glycerol and double distilled water and keep the seeds at -80℃.

3.1.3 Test

We verified that our plasmid was successfully transferred to Synechocystis sp. PCC 6803 by colony PCR. The specific method is to pick the selected single colony of Synechocystis sp. PCC 6803 and delineat for expanded culture on plates. After more colonies grew up, we pick them into liquid BG11 medium to get enough bacteria solution and extract the Synechocystis sp. PCC 6803 genome. And conduct PCR verification after the bacteria is amplified for avoiding PCR failure caused by the engineering bacterium concentration is too low, or PCR success but strain amplification failure cases. Fig 10. The PCR of pLactate, omcs,and presistance6803 colonies all had bright and correct bands, which proved that the transformation was successful.

After colony PCR identified bright and correct bands, we preserved them for expanded culture and induction of expression. We examined the growth curves of wild-type Synechocystis sp. PCC 6803 and Synechocystis sp. PCC 6803(omcs) in BG11 medium. Compared to Synechocystis sp. PCC 6803, Synechocystis sp. PCC 6803(omcs) exhibited a significant improvement in cell concentration at all stages, and there was no significant decrease in growth rate. This suggests that omcs can enhance the photosynthetic efficiency of PS1 by increasing ATP synthesis, which promots better growth of cells. It proved that the genes we transferred into Synechocystis sp. PCC 6803 were finally effectively expressed.

At the same time, we detected the amount of lactate produced by wild-type Synechocystis sp. PCC 6803 and Synechocystis sp. PCC 6803 engineering bacteria pLactate. Comparing the data of wild-type and engineered bacteria pLactate, we found that the amount of expression products of Synechocystis sp. PCC 6803 engineering bacteria pLactate were higher than those of wild-type Synechocystis sp. PCC 6803, demonstrating that the strain we have built works as expected.

3.2 Shewanella oneidensis MR-1

3.2.1 Design

We hope to verify that the strain we built worked as efficiently as expected by measuring the amount of the expression product NADH/NAD+. At the same time, we compared whether the ability to generate electricity of S. oneidensis MR-1 transferred with the plasmid we constructed was stronger than that of wild-type S. oneidensis MR-1 by measuring the efficiency of electricity production from S. oneidensis MR-1.

3.2.2 Build

We culture the strain containing the correct plasmid and extract the plasmid to transfer into E. coli WM3064. Subsequently, we transferred the plasmid we constructed into S. oneidensis MR-1 by conjugation between E. coli WM3064 and S. oneidensis MR-1. The experimental operation is: we inoculated an equal and appropriate amount of E. coli WM3064 with S. oneidensis MR-1 into 1 ml liquid LB medium containing 0.019 g/mL DAP and 50 μg/mL kanamycin for one hour, and finally inoculated the mixed bacteria solution on LB plates containing 50 μg/mL / mL of kanamycin. When single colonies of S. oneidensis MR-1 appeared on the surface of solid media, we singled out the single colonies for PCR validation. After observing the correct bands, we mixed 800 μl of S. oneidensis MR-1 solution with 800 μl of 1:1 mixture of glycerol and double distilled water and keep the seeds at -80℃.

3.2.3 Test

We verified that our plasmid was successfully transferred to S. oneidensis MR-1 by colony PCR. The specific method is to culture the selected single colony of S. oneidensis MR-1 in a shake flask containing 1ml liquid LB with Kana, and conduct PCR verification after the bacteria is amplified for avoiding PCR failure caused by the engineering bacterium concentration is too low, or PCR success but strain amplification failure cases. Fig 14. The PCR of pElectricity, ycel-pncB, nadE-nadD-nadM, presistanceMR-1 colonies all had bright and correct bands, which proved that the transformation was successful.

After colony PCR identified bright and correct bands, we preserved them for expanded culture and induction of expression. We measured the amount of NADH/NAD+ produced by wild-type S. oneidensis MR-1 and S. oneidensis MR-1 with plasmid ycel-pncB or nadE-nadD-nadM. All bacteria were cultured in LB medium with Kana. When the OD₆₀₀ of medium reached 0.6-0.8, we added 0.5mM IPTG to intrudce the target gene expression. Culture overnignt after introduing, and extract NAD(H/+) by NAD(H/+) extraction kit. Compared to the wild type,S. oneidensis MR-1(ycel-pncB) showed a 22.23% increase in NADH/NAD+ ratio, while the total amount of NADH/NAD+ inS. oneidensis MR-1(nadD-nadE-nadM) increased by 27.34%. This indicates that S. oneidensis MR-1(ycel-pncB) may contribute to the accelerated transport of niacin and nicotinamide for intracellular NADH synthesis, while S. oneidensis MR-1(nadD-nadE-nadM) facilitates more efficient electron transfer, proving that the working efficiency of the strain we constructed achieved our expectations.

At the same time, we tested the electricity production of wild-type S. oneidensis MR-1 and S. oneidensis MR-1 with plasmid ycel-pncB or nadE-nadD-nadM. Comparing the electricity production of the two, we found that the voltage of the battery constructed by S. oneidensis MR-1 engineering bacteria ycel-pncB and nadE-nadD-nadM was higher than that of the battery constructed by wild-type S. oneidensis MR-1.