Team:HK SSC/Engineering
Engineering Success
The effect of manganese concentration in BG-11 on cell growth
Design
To optimize the growth of S.elongatus UTEX 2973, we decided to investigate the BG-11 growth medium, the immediate environment the cells depend on. The original BG-11 medium, as published by Thermofisher, contains a number of trace metals, one of them being manganese. Manganese is a crucial trace metal required for the functioning of the photosystem II in cyanobacteria. The growth of cyanobacteria can fluctuate greatly based on change in concentration of manganese, showing its sensitivity to the trace metal. Grasping the precise concentration of manganese in BG-11 for optimal growth of S.elongatus UTEX 2973 enables us to maximize the use of the resources used to grow the cells. Therefore, we decided to grow cells in setups of BG-11 with different concentrations of manganese and compare their cell growth.
Build
The concentrations of the manganese used in each setup was relative to the original concentration in BG-11 medium. Cells were grown in setups with BG-11 with 0.1 times, 0.5 times, 1 times, 1.5 times and 2 times of the original concentration of manganese respectively. Their OD730 was recorded daily for five days. The setups were placed in a 30ºC incubator with LED lights shining in cycles of on for two hours then off for one hour.
Test
All setups showed increase in OD730 over the course of 5 days. Generally, the lower the concentration of manganese the higher the OD730. Cells grown in the setup with 0.1 times the original manganese concentration of BG-11 showed significantly higher cell growth than the other setups. A slight decrease in 0D730 was observed on the fifth day in the setup with 2 times the original manganese concentration.
Learn
It can be seen that BG-11 medium with 0.1 times its original manganese concentration led to consistently higher S.elongatus UTEX 2973 cell growth. This shows that a low concentration of 0 to 0.0009 mmol dm-3 manganese is sufficient for optimal cell growth, any higher than that will likely cause cell death. We will adjust our BG-11 medium according to this result to enhance the growth of S.elongatus UTEX 2973 for our project.
The Choice of using the INTEGRATE system
Design
As genomic integration is preferred over plasmid-based expression due to it being more stable and predictable, we chose the INTEGRATE system enables us to insert genes directly into the chromosome of S.elongatus UTEX 2973. We prefer using this CRISPR-transposon genome editing technique over homology directed repair (HDR) methods as it is simpler due to the lack of need for homology arms1. The INTEGRATE system is also a more suitable CRISPR tool for S.elongatus UTEX 2973 compared to CRISPR-Cas9 systems since high expression levels of Cas9 proteins in S.elongatus UTEX 2973 induce toxicity and cell death. Transient expression of Cas9 achieves genomic engineering in S.elongatus UTEX 2973, but compared to the streamlined INTEGRATE system, it is less predictable2.
Build
The original pSPIN plasmid for INTEGRATE system by Vo et al. (2020) is for replication and expression in E.coli, so we plan to obtain the sequence coding for necessary genome editing proteins from pSPIN by restriction enzyme digestion. This sequence contains a site for users to clone their genes of interest as a donor DNA to be inserted into the genome of expression host organism. This sequence has been registered as BBa_K3776022 and users can clone it into backbone vectors they wish to use and clone their genes of interest at the Donor DNA site with reference to Vo et al. (2020). In our project, we will use standard cloning techniques to ligate in AK, DavB and DavA, and CF3BD as the donor DNA in this part then clone the part with these genes into our shuttle vector panS-loxP-MCS. The shuttle vector with the INTEGRATE system with DavB, DavA and AK as the donor DNA is registered as BBa_K3776020. The shuttle vector with the INTEGRATE system with CF3BD as the donor DNA is registered as BBa_K3776021.
Test
We were able to transform and clone E.coli DHα cells with pSPIN for plasmid preparation. E.coli DHα cells after transformation with pSPIN showed resistance to streptomycin.
Due to limitations using the laboratory due to the pandemic, we have not been able to characterize the INTEGRATE system functioning in S.elongatus UTEX 2973 this year. We will continue the work in the second phase of our project.
References:
- Vo, P. L. H., Ronda, C., Klompe, S. E., Chen, E. E., Acree, C., Wang, H. H., & Sternberg, S. H. (2020). CRISPR RNA-guided integrases for high-efficiency, multiplexed bacterial genome engineering. Nature Biotechnology, 39(4), 480–489. https://doi.org/10.1038/s41587-020-00745-y
- Wendt, K. E., Ungerer, J., Cobb, R. E., Zhao, H., & Pakrasi, H. B. (2016). CRISPR/Cas9 mediated targeted mutagenesis of the fast growing cyanobacterium Synechococcus elongatus UTEX 2973. Microbial Cell Factories, 15(1). https://doi.org/10.1186/s12934-016-0514-7