In the future, iGEMers can get the following inspirations and inspirations from our projects:
1. “Transport-chelate- redox” system:
Suppose you find that it is difficult for cells to grow in harsh environments, such as Synechocystis, in water with a high cadmium concentration. In that case, you may wish that Synechocystis can survive and be fit in it. You can improve the three functions of Synechocystis--transport, chelate and redox the cadmium ions. Therefore, our team has picked out the protein of transport, chelation and core-redox enzymes, which express highly in plants or yeasts. After transporting many cadmium ions into cells, heavy metal ions can be stored in cells by chelating protein. In the meantime, the expression level of redox enzymes can be increased to remove intracellular reactive oxygen species (ROS) and improve the growth rate of Synechocystis under the stress of cadmium ions. Also, for the high expression of functional proteins, our team has carried out codon optimization according to the codon preference of Synechocystis. We have constructed a synthetic biobrick which could be used by future teams.
As an additional strategy, We used the SmtA protein constructed by Groningen's team in 2009 (http://parts.igem.org/Part:BBa_K190021)to adsorb cadmium ions from outside the cell. We have successfully expressed protein in E. coli DH5α and supplemented experimental data to demonstrate that SmtA can Improve cadmium tolerance of E. coli respectively.（Figure a、b）
Figure a：Western blotting results indicating the successful expression in E.Coli
Figure b：Growth curve after successful protein expression
In theory, cadmium and zinc are elements of the same family. Natural cadmium often co-existing with zinc, cadmium pollution occurs following collecting the zinc ore. And therefore, it may be envisaged that similar mechanisms can also be used to remove mixed heavy metal pollution and extend the use of Synechocystis. If you want to use algae to remove other metal ions, please try it!
2. “Toxin-antitoxin” suicided system:
In our study, we take biological security very seriously. Suppose you want to ensure that the organisms modified in the lab will not cause environmental pollution or harm the environment. In that case, you can use our “toxin-antitoxin” suicide system! We have sorted out a light control system. Inhibition of toxin expression under blue light resulted in the average growth of algae. When the blue light is gone, the toxin is highly expressed, and the algae will die. The advantage of the light control system is that it’s simple to control the death of Synechocystis. by control the expression of toxin genes as a switch.
3. The experience in culturing Synechocystis:
(1) MAKING THE OD VALUE ABOVE 0.3! When culturing Synechocystis, a low concentration of Synechocystis leads them dead by the blue light.
(2) IT’S NO NEED TO PLACE Synechocystis IN Shaker FOR WHOLE DAY.
(3) Synechocystis is best grown in conical flasks, not centrifuge tubes
(4) it is better to grow 50ml bottles with a 30ml-50ml medium.