Section 1 : Introduction

Our project aimed to enable Synechocystis sp. PCC 6803 to produce lactic acid at a high efficiency, enhance the electricity producing capacity of S. oneidensis MR-1, as well as improve the resistance of both bacteria. In order to achieve these goals, we construct mainly 4 plasmids: pLactate and pResistence6803 for Synechocystis sp. PCC 6803, pElectricity and pResistance MR-1 for S. oneidensis MR-1. Besides 4 main plasmids, we also constructed 3 plasmids in order to test the function of genes more conveniently.

We transferred them into Synechocystis sp. PCC 6803 and S. oneidensis MR-1 for expressing the proteins we needed. At the same time, in order to verify the efficiency of electricity production, we also carried out and verified our project from all aspects.

Section 2 : Plasmid amplification and reconstruction

We first transformed the plasmids containing designed sequence synthesized by the company into Escherichia coli Top10 for amplification, so we have enough amount of plasmid to carry out subsequent experiments. Then colony PCR showed a successful transformation, and required plasmids were extracted for the next experiment.

We then obtained target fragments by means of PCR, and then constructed them into the plasmids we need.

2.1 Plasmids for Synechocystis sp. PCC 6803

We have 2 plasmids, pLactate and pResistance6803 for Synechocystis sp. PCC 6803. We verified the size of each plasmids as well as all the fragments involved in constructing the plasmids. In addition to pLactate and pResistance6803, we also constructed plasmid Omcs to ensure the function of it individually.

pLactate includes LdhA enabling Synechocystis sp. PCC 6803 to produce lactic acid by means of photosynthesis, Lldp to transform intracellular lactate to the extracellular as well as Omcs improving the efficiency of electricity transformation.

The bands of Pcpc560-ldhA-lldP-Tpsbc-Ppsba2-omcs-Tpsbc(5000+bp) from PCR are identical to the theoretical lengths of 5479bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these plasmids had successfully been obtained.

The bands of Pcpc560-ldhA-lldP-Tpsbc(3000+bp) from PCR are identical to the theoretical lengths of 3643bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these plasmids had successfully been obtained.

The bands of Ppsba2-omcs-Tpsbc(almost 2000bp) from PCR are identical to the theoretical lengths of 1866bp(from pLactate) and 1820bp(from Omcs) estimated by the designed primer locations (promoter to terminator), which could demonstrate that these plasmids had been successfully obtained.

The bands of PUC57-slr0168(vector)(almost 5000bp) from PCR are identical to the theoretical lengths of 4727bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these plasmids had been successfully obtained.

All the results above showed us a success of the construction of pLactate and Omcs.

pResistance6803 is constructed to enhance the resistance of Synechocystis sp. PCC 6803 under adversities, especially the antioxidant capacity and the ability to protect DNA from damages.

The bands of PrbcL-dsup-Tpsbc-PrbcL-gshA-gshB-Tpsbc(almost 5000bp) from PCR are identical to the theoretical lengths of 4746 estimated by the designed primer locations (promoter to terminator), which could demonstrate that these plasmids had been successfully obtained.

The bands of PrbcL-dsup-Tpsbc (almost 2000bp) from PCR are identical to the theoretical lengths of 1848 estimated by the designed primer locations (promoter to terminator), which could demonstrate that these plasmids had been successfully obtained.

The bands of PrbcL-gshA-gshB-Tpsbc(almost 3000bp) from PCR are identical to the theoretical lengths of 2889 estimated by the designed primer locations (promoter to terminator), which could demonstrate that these plasmids had been successfully obtained.

The bands of PUC57-slr2030(almost 5000bp) from PCR are identical to the theoretical lengths of 4633 estimated by the designed primer locations (promoter to terminator), which could demonstrate that these plasmids had been successfully obtained.

All the results above showed us a success of the construction of pResistance6803.

2.2 Plasmids for S. oneidensis MR-1

We have 2 plasmids, pElectrcity and pResistance MR-1 for S. oneidensis MR-1. We verified the size of each plasmids as well as all the fragments involved in constructing the plasmids . Besides pElectricity and pResistanceMR-1, we also constructed plasmids to ensure the function of ycel-pncB and nadE-nadD-nadM individually.

pElectricty is constructed with ycel-pncB and nadE-nadD-nadM, both can elevate the electricity production rate of S. oneidensis MR-1.

The bands of Ptac-ycel-pncB-TrrnB T1-Ptac-nadE-nadD-nadM-TrrnB T1(5000+bp) from PCR are identical to the theoretical lengths of 5736 estimated by the designed primer locations (promoter to terminator), which could demonstrate that these plasmids had been successfully obtained.

The bands of Ptac-ycel-pncB-TrrnB T1(2500+bp) and Ptac-nadE-nadD-nadM-TrrnB T1(about3000 bp) from PCR are identical to the theoretical lengths of 2624bp and 2820bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these plasmids had been successfully obtained.

The bands of Ycel-PncB(about 2500bp) from PCR are identical to the theoretical lengths of 2622bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these plasmids had been successfully obtained.

The bands of Ptac-nadE-nadD-nadM-TrrnB T1 (about 3000bp) from PCR are identical to the theoretical lengths of 2820bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these plasmids had been successfully obtained.

The band of PYYDT(vector) (5000+bp) from PCR is identical to the theoretical lengths of 5886bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these plasmids had been successfully obtained.

All the results above showed us a success of the construction of pElecteicity, ycel-pncB and nadE-nadD-nadM.

pResistance MR-1 is constructed with dsup-sodA and oprf, which can lower the level of ROS in S. oneidensis MR-1 and avoid DNA damages, and the latter can assist in the entry and exit of riboflavin and improves the efficiency of electron transfer

The bands of Ptac-dsup-sodA-oprf-TrrnB T1(3000+bp) from PCR are identical to the theoretical lengths of 3291bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these fragments had been successfully obtained.

The bands of Ptac-dsup-sodA-oprf-TrrnB T1(3000+bp) from PCR are identical to the theoretical lengths of 3291bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these fragments had been successfully obtained.

The bands of Ptac-oprf-TrrnB T1(1000+bp) from PCR are identical to the theoretical lengths of 1135bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these fragments had been successfully obtained.

The results above could demonstrate that these plasmids are correctly constructed, so we can transform pLactate, pResistence6803,and omcs into Synechocystis sp. PCC 6803, while pElectricity, ycel-pncB, nadD-nadE-nadM and pResistance MR-1into S. oneidensis MR-1 for verification.

Section 3 :Strain construction

We have successfully constructed all the target plasmids needed in the four loops, and this step really facilitates our subsequent experiments. At the same time, in order to facilitate the measurement of the function of some elements, we separately constructed plasmids (omcs, ycel-pncB, nadD-nadE-nadM) for some essential elements.

All the bands are identical to the theoretical sizes, demonstrating that these plasimds had been successfully transformed and verified.The results are as follows: PLactate: Pcpc560 - ldhA - lldP - Tpsbc - Ppsba2 - omcs -Tpsbc about 5400 bp , pResistance6803: PrbcL - dsup - Tpsbc - PrbcL - gshA - gshB -Tpsbc about 4100 bp , omcs: Ppsba2-omcs-Tpsbc about 1800bp, pElectricty:Ptac-ycel-pncB-TrrnB T1-Ptac-nadD-nadE-nadM-TrrNB T1 about 5700bp, pResistance MR-1: Ptac-dsup-soda-oprf-TrrnB T1 about 3200bp , ycel-pncB: Ptac-Ycel-PNCB-TrrnB T1 about 2500bp, nadD-nadE-nadM: Ptac-nadD-nadE-nadM-TrrNB T1 about 2800bp

The theoretical length of 5461bp, 4107bp,1809bp, 5666bp, 3229bp, 2564bp and 2820bp estimated by the design primers location (promoter to terminator) was the same, indicating that we successfully obtained the plasmid we needed.

The successful plasmids transformed into Synechocystis sp. PCC 6803 and S.oneidensis MR-1 was verified by colony PCR.

Section 4 : Expression Validation

4.1 Synechocystis sp. PCC 6803

4.1.1 Growth curve of Synechocystis sp. PCC 6803

We used BG11 to culture the engineered bacteria Synechocystis sp. PCC 6803(omcs) and wild type respectively, and measured the OD₇₃₀ every 24 hours. A growth curve is obtained as follows.

The results showed that 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 PSI by increasing ATP synthesis, which promots better growth of cells

4.1.2 Lactate production verification

We want to co-culture Synechocystis sp. PCC 6803 and S. oneidensis MR-1. The nutrient of S.oneidensis MR-1 is lactate produced by Synechocystis sp. PCC 6803. We tried to determine the production of lactate by HPLC to test the feasibility of the co-culture method.

We first measured the standard curve of lactate concentration and determined its retention time. The Synechocystis PCC6803(pLactate) cells were broken by freeze-thaw method, and the supernatant after centrifugation was taken for high performance liquid chromatography (HPLC).

A peak with almost the same retention time which is 3.932 min as the lactate standard substance was observed on the spectrum, which confirmed the successful expression of lactate in engineered bacteria. The peak area was compared with the standard curve, which can be seen that the lactate yield of our engineering Synechocystis sp. PCC 6803 was 7.26mM, indicating that the gene we introduced could produce lactic acid stably and provide nutrition for S. oneidensis MR-1.

This result indicated the successful expression of the imported LdhA and LldP genes, and verified the feasibility of the co-culture system.

4.2 S. oneidensis MR-1

4.2.1 Determination of NADH/NAD+ concentration

To verify normal gene expression in S. oneidensis MR-1, we measured the NADH/NAD+ content of wild type and engineered strains ycel-pncB and nadD-nadE-nadM, respectively.

Compared to the wild type, S. oneidensis MR-1(ycel-pncB) showed a 22.23% increase in NAD(H/+) amount, while the total amount of NAD(H/+) in S. 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.

4.2.2 Verification of electricity production of S. oneidensis MR-1

We built a microbial fuel cell device to test the electricity-producing performance ofS. oneidensis MR-1.

We inoculated S. oneidensis MR-1 cultured overnight in 100 mL LB medium at 30℃ and 200 rpm to OD₆₀₀ to 0.6-0.8. The cultured cells were collected by centrifuge and re-suspended in 140 mL anoditic solution. 50 μg/mL kana antibiotics and 18mM lactate were added to maintain constant culture conditions and 0.1 mM IPTG inducer to initiate gene expression. The two electrode chambers of MFC are separated by proton exchange membrane, using carbon cloth as cathode and anode electrodes, and then adding 140mL cathode solution to the cathode chamber, using 2kΩ external resistance to close the external circuit.

The specific battery structure of a complete MFC is as follows

1) Testing the MFC

After construction the MFC, we use a digital multimeter to measure the output voltage of microbial fuel cell.

We measured the voltage of S. oneidensis MR-1(ycel-pncB), S. oneidensis MR-1(nadD-nadE-nadM) and compared them with the voltage of S. oneidensis MR-1. Measure a set of voltages every 6 hours and compare as follows.

The results showed that S. oneidensis MR-1(ycel-pncB) exhibited a significantly higher power output than the wild type before 30 hours, but experienced a noticeable decrease after 30 hours. It is speculated that this could be attributed to the ability ofS. oneidensis MR-1(ycel-pncB) to accelerate the transport of niacin and nicotinamide for intracellular NADH synthesis, resulting in a higher power output before 30 hours.

However, due to the absence of exogenous niacin and nicotinamide supplementation, the availability of NADH synthesis precursors decreases, leading to a decline in intracellular NADH concentration and a slower electron transfer rate, ultimately resulting in reduced power generation efficiency.

In contrast, nadD-nadE-nadM exhibited significantly higher discharge peak and prolonged high-efficiency discharge duration compared to the wild type. The highest out put voltage was up to 150.7 mV, with a 42.32% increase in the hightest power output .

2) Exogenous electron carrier

In our search for relevant papers, we learned that exogenous electron carrier such as photosensitivities and riboflavin contribute to extracellular electron transfer, thereby improving the efficiency of electricity generation. Therefore, we investigated the effect of addition of photosensitizers (5 μM Eosin Y, 5 μM Fluorescein) and 40 μM riboflavin on electricity production in S. oneidensis MR-1.

We use S. oneidensis MR-1(nadD-nadE-nadM) to try getting the highest voltage output by adding exogenous electron carrier including riboflavin and different photosensitizers. We constructed the MFC device and observed that riboflavin significantly increased the peak power output by 5.56 times after 20 hours with the highest voltage at 668mV, while the other exogenous electron carrier showed no significant change. This could be attributed to the optimization of electron carriers and enhanced electron transfer efficiency after the addition of riboflavin.

The results show that riboflavin is helpful in generating electricity.And considering the low cost of riboflavin, it can be applied in subsequent industrial production.

3) Practical application

In order to verify the practical application effect of the microbattery, we connected the microbattery containing the gene pathways of S. oneidensis MR-1, S. oneidensis MR-1(ycel-pncB), S. oneidensis MR-1(nadD-nadE-nadM)with a red LED bulb , respectively.

The results showed that the brightness of LED bulb did not change significantly after being connected with the S. oneidensis MR-1 micro battery, indicating that the S. oneidensis MR-1 micro battery did not reach the operating voltage of LED bulb, while the red LED bulb in the engineering bacteria group could be used normally and emit bright red light. The hightest voltage could be up to 2.1V

The results showed that the gene we transferred significantly improved the electricity generation capacity of S. oneidensis MR-1, which can actually provide electrical energy for some electrical appliances.

4.3 Co-culture system

4.3.1 Co-culture growth

We attempted to combine the BG11 medium required for Synechocystis sp. PCC 6803 culture with the M9 buffer used for S. oneidensis MR-1 to generate electricity and configured MBG11 medium to achieve the growth of two kinds of bacteria through the same medium, so as to prepare for the subsequent continuous culture experiment.

We used MBG11 culture medium to culture Synechocystis sp. PCC 6803 and S. oneidensis MR-1 respectively, and measured OD values of Synechocystis sp. PCC 6803 culture medium at 730nm and S. oneidensis MR-1 culture medium at 600nm. The OD value is measured every 24 hours, and a growth curve is obtained as follows. It can be seen that MBG11 culture can make the normal growth of Synechocystis.

However, at the same time, we found that S. oneidensis MR-1 could grow at present, but the growth rate was slow. When the bacteria reached the stable stage, the concentration of the bacteria was not high, and the growth situation was far less than that cultured in LB medium. We analyzed that the reason for this result might be the lack of nutrients.

At the same time, we also noticed that when M9 buffer was used to generate electricity for a long time, the bacterial concentration in the device was almost unchanged, indicating that in a low-nutrient environment, S. oneidensis MR-1 could only maintain survival and generate electricity but could not grow and multiply.

In response to this situation. We will further improve the medium system in the future, and at the same time increase the concentration of S. oneidensis MR-1 in LB medium in advance and add MBG11 medium to produce electricity.

4.3.2 Co-culture to produce electricity

We used MBG11 medium to measure the electric production performance of Shiwanella in logarithmic growth phase. The results are as follows, indicating that it can produce electricity normally in MBG11 medium containing lactic acid, and further prove the feasibility of co-culture scheme.