Safety

Safety

While the prospects for the application of genetically modified cyanobacteria are exciting, biosafety concerns have been raised regarding the accidental release of these modified organisms into the natural environment. Particularly given that cyanobacteria are robust organisms with high adaptability, the consequences of their presence in natural ecosystems are unpredictable. Fully aware of this and to ensure the safety of all our team members, our team took all the necessary precautions to ensure we do not cause any environmental or personal harm.

Chassis Safety ///

We utilized E.coli DH5α, E.coli BL21 (DE3) and Synechocystis sp. PCC 6803 to ensure the safety of our project. These microorganisms are classified in the risk group 1; they present low risk for human safety and the environment.

Parts Safety ///

To produce the different fragrance molecules, we introduced 10 genes into Cyanobacteria. Before building the plasmids, we checked the origin of the genes we wanted to use. The genes all belong to the white list provided by iGEM since they all come from the genome of plants.

Although the fragrance molecules we desire to produce in cyanobacteria (pinene, limonene, bisabolene, farnesene, santalene, and santalol) carry some degree of risk, they are found in various essential oils and are widely used in aromatherapy. This suggests that they do not pose serious risks to humans when consumed in low quantities. Additionally, our cyanobacteria is unable to produce these fragrance molecules in quantities large enough to constitute serious risks.

Suicidal System ///

Our project consists in using microorganisms modified by the introduction of heterologous genes. Due to that many cyanobacteria species are naturally transformable, accidental release of engineered cyanobacteria increases the possibility of the spread of synthesized genes into the natural cyanobacteria population via horizontal gene transfer. To guarantee that none of our modified cyanobacteria are able to survive in natural environments, it was necessary for us to develop a suicidal system in Synechocistis sp. PCC 6803.

inspired by the molecular engineering work carried out by Zhou et. al and to ensure the safety of our project, we have built an active containment system in Synechocystis sp. PCC 6803 with a controllable “kill switch” that causes cell death. We introduced a controllable metal ion induced biocontainment system consisting of an Fe3+ induced toxin-antitoxin system using the broad host range replicative vector pPMQAK1 that is based on the replicon RSF1010. In the type II TA system, the anti-toxin protein neutralizes the toxin via direct protein reaction. The toxin, which restrains the replication of DNA, digests mRNA and inhibits the synthesis of proteins, will only be expressed when there is no of Fe³⁺ ion present, therefore leading to a programmed suicide in environments lacking Fe³⁺. Although the growth of wild type Synechocystis sp. PCC 6803 is repressed in growth mediums lacking Fe³⁺ ion, this biocontainment system assures that none of the escaped engineered cyanobacteria survive in natural environments. Compared to other strong induction and low leakage promoters, the Fe³⁺ repressed promoter has a clear advantage due to that there is no need for additional inducers. Fe³⁺ ion deficiency in marine and fresh water allows the “kill switch” in the modified cyanobacteria to be turned on as soon as it reaches natural environments [3].

Figure 1: Diagram of type II TA system.

We constructed four different plasmids using two toxin-antitoxin pairs, one light inducible promoter, and two different Fe³⁺ repressed promoters. Antitoxins were expressed using the light inducible promoter PpsbA2 from Synechocystis sp. PCC 6803, which is active in the cyanobacteria under normal cultivation conditions. Toxins, instead, were expressed using the Fe³⁺ ion repressed promoter PisiA6803 and PisiA7942 from Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 respectively. The two different toxin-antitoxin pairs we used are sepA1/sepT1 (PemK-like system) and sepA2/sepT2 (VapC-like system) from Synechococcus elongatus PCC 7942. Finally, we used BBa_B0015 as the terminator.

• BS1: PpsbA2-sepA1-PisiA6803-sepT1
• BS2: PpsbA2-sepA2-PisiA6803-sepT2
• BS3: PpsbA2-sepA1-PisiA7942-sepT1
• BS4: PpsbA2-sepA2-PisiA7942-sepT2

Figure 2: Diagram of the biosafety plasmid we constructed.

We applied the same approach as Zhou et. al, which tested the system in Synechococcus elongatus PCC 7942 and Synechococcus elongatus UTEX 2973; however, there are no previous studies that have evaluated this biocontainment system in Sycechocystis sp. PCC 6803.

Results ///

Once we constructed the biocontainment system plasmids, we transformed them into E. coli DH5α and performed colony PCR on the monocultures. Below are the colony PCR results.

Figure 3: BS1, BS2, BS3, BS4 colony PCR fel electrophoresis results in E. coli.

We then selected the successful ones and amplified them to transform them into Synechocystic sp. PCC 6803. Below are the colony PCR results.

Figure 4: BS1, BS2, BS3, BS4 colony PCR fel electrophoresis results in Syn. sp. PCC 6803

After successfully transforming our constructed plasmid into Synechocystis sp. PCC 6803, we assessed the effectiveness of the suicidal system by growing the engineered cyanobacteria on agar plates with and without iron. We adjusted the samples to OD750=0.1 and further diluted them to 0.01, 0.001 and 0.0001. We dropped the diluted samples onto BG-11 agar plates with Fe³⁺ ions and BG-11 agar plates without Fe³⁺ ions. As a control group, we also dropped engineered Synechocystis sp. PCC 6803 containing our constructed plasmid, αPS, on both agar plates with and without Fe³⁺

Figure 5: Biosafety samples on BG-11 agar plates with and without Fe³⁺ ion after 1 week of cultivation.

Through our first attempt of testing the effectiveness of the biocontainment system in Synechocystis sp. PCC 6803 we were unable to get clear results of whether the cyanobacteria with the system died on the agar plate without Fe³⁺, or the concentration was too low for us to see it. As can be seen on the picture of both agar plates, all cyanobacteria were able to grow normally on the agar plate with Fe³⁺ ion and none of the cyanobacteria with the biocontainment system can be seen on the agar plate without Fe³⁺ ion. However, our control group, αPS, did not grow either. We then realized that cyanobacteria growth is repressed in environments lacking Fe³⁺ (4), thus the cell concentration must be very low and appears as if it has not grown.

We speculate that BG-11 plate cultures may not be suitable for assessing Fe³⁺ deprivation status in cyanobacteria, so we decided to test the biocontainment system in Synechocystis sp. PCC 6803 by cultivating the cyanobacteria in liquid BG-11 medium with and without Fe³⁺ ions. Since cyanobacteria grows faster in liquid BG-11 medium, we hoped to see more apparent results from the control group growing in the Fe³⁺ -deficient medium.

Figure 6: Synechocystis sp. PCC 6803 containing the BS plasmids cultivated in liquid Fe³⁺ -deficient BG-11 medium.

After 5 days of cultivation, we got the above results. Synechocystis sp. PCC 6803 contianing the plasmids BS3 and BS4 were not able to grow in Fe³⁺ -deficient BG-11 medium. However, we did not get the desired results with those containing BS1 and BS2 plasmids. These results suggest that the promoter PisiA7942 potentially performs better in Synechocystis sp. PCC 6803 compared to PisiA6803.

Additionally, we cultivated the cyanobacteria with the biocontainment system in our bioreactor.

Figure 7: The growth of cyanobacteria with the biocontainment system (BS3) in our bioreactor.

Figure 8: Day 1 of the cyanobacteria with the biocontainment system (BS3) in our bioreactor

Figure 9: Day 5 of the cyanobacteria with the biocontainment system (BS3) in our bioreactor

From the pictures, it can be clearly seen that the cyanobacteria concentration in the bioreactor decreased significantly after 5 days of cultivation in BG-11 medium lacking Fe³⁺.

Lab Safety ///

Prior to starting any actual experiments, our advisors spent two weeks giving us detailed lessons on lab safety and guiding us through the basic experimental procedures. To ensure the safety of our team members in the lab, we conducted all experiments under the supervision of at least one of our instructors. In the lab, we also have a special hazardous area in which we perform all electrophoresis and manipulate the nucleic acid dye. We strictly prohibited food to be brought in the lab and wore appropriate clothing, lab coat and gloves for experiments at all times. After experiments, we cleaned up everything and deposited waste accordingly. Additionally, we participated in a fire drill to further ensure the safety of everyone in the lab.

Unfortunately, the COVID-19 pandemic was still running in China at the beginning of our project. To ensure the safety of all members, we held our weekly meetings online when necessary, and entry into the lab building was strictly controlled.