Synthetic biology is based on standard parts, and characterizing a specific part can save future users' time from researching each necessary component when building their systems. Inter-lab works can help characterize a part more comprehensively by using different materials and methods. This year, we measured the new parts we created including:
Our measurement provides abundant experimental data for these parts and provides the basis for promoting the engineering application of these parts.
Result
TLS: Measurement of mobility for sequences of different lengths
After the initial determination that TLS can carry RNA movement, we hope to more specifically characterize the ability of different TLS to carry RNA sequences of different lengths. Given that TLS moves through plants in the form of RNA complexes, real-time fluorescent quantitative PCR (RT-qPCR) was used to determine the amount of destination RNA at different distances to quantify mobility. We used the relative expression of RNA 1 cm from the site to characterize TLS mobility.
At the same time, to quantify the ability of different TLS carrying RNA sequences of different lengths, we selected three TLS(TLS(Ara.Met.full), TLS(Ara.Met.ΔΔt), TLS(Nico.Met.full)) and measured their ability to carry large fragments (Ruby, 4 kb) and small fragments (GFP, 700 bp).
The results show that different TLS exhibit different capabilities for carrying fragments of different length(Fig.1). The measured data can guide our further modification and selection of TLS. For example, while the migration ability of all three TLS was significantly reduced in carrying longer sequences (about 50-fold decrease in RNA relative expression at 1cm), the relative expression level of RNA at 1cm of TLS (Ara.Met.ΔΔt) was only reduced by about 20-fold(Fig.1 b). This suggests that appropriate deletion of stem rings may facilitate TLS's ability to carry long fragments.
RNA dependent RNA polymerase: Measurement of self-replication ability at different levels
As for RNA-dependent RNA polymerase, its self-replication function has been verified at different levels. After seeing the phenotype under a fluorescence microscope, we used image processing software to measure the fluorescence intensity of the images at the same exposure intensity. At the same time, protein and RNA were extracted from the transfected leaf tissues. At the protein level, fluorescent enzyme labeling instruments are used to detect the content of the target protein GFP in the protein extract. At the RNA level, RT-qPCR was used to detect the relative expression of RNA in the extracts.
At the phenotypic level, we preliminarily confirmed the self-replication function of RdRp, and found that different subgenomic promoters can achieve different fluorescence intensities.Compared with the control group, the fluorescence intensity of the subPromoter MP group expressing RdRp can reach 2.5 times, while the fluorescence intensity of the subPromoter CP group can reach 3.5 times(Fig.2 a).
The results of protein level were basically consistent with phenotype.Tissue expressing GFP from subPromoter MP produced a mean value of 19.13 relative fluorescence units., 4-fold more GFP in comparison to control group that produced a mean of 4.87 units. While that from subPromoter CP produced a mean value of 30.07 relative fluorescence units., 6-fold more GFP in comparison to control group(Fig.2 b).
The RNA level more directly characterizes RdRp's self-replicating ability and the transcriptional effects of two different subgenomic promoters. After normalization with the Ct value of control group, the average relative expression of the subPromoter MP group was about 4 and that of subPromoter CP was about 10(Fig.2 c).
Biosafety: Measurement of a new promoter strength and photoinduced suicide protein function
Agrobacterium tumefaciens promoter
Since the studies on Agrobacterium tumefaciens chassis in the iGEM community are far less than those on commonly used chassis such as Escherichia coli, we wanted to explore the available promoters in Agrobacterium and try to express proteins originally expressed in Escherichia coli in Agrobacterium.
We first choose 50Spro(BBa_K4628022), which is responsible for encoding the 50S ribosomal protein L28 in the Agrobacterium tumefaciens C58 genome. We believe that it is a relatively strong constitutive promoter.
In addition to this, we measured Pvbp2(BBa_K4628022), an inducible promoters. It controls the expression of VirD2-Binding Protein in Agrobacterium tumefaciens, which is used to control T-DNA transfer.
In our experiments, we used green fluorescent protein (GFPuv, BBa_M45116) as a reporter gene to characterize the promoter. The expression of this gene was observed using a multifunctional imager.
We used the gray value to represent fluorescence intensity, demonstrating that both of them could initiate gene expression, and 50Spro was significantly stronger than Pvbp2.
Phototoxic proteins
KillerRed and miniSOG proteins, which have been measured in Escherichia coli, were selected for characterization. One of them, miniSOG, was codon-optimized to make it more suitable for expression in Agrobacterium tumefaciens. Under the control of 50Spro, both proteins can be expressed and control Agrobacterium tumefaciens suicide, and miniSOG can be seen as obvious green fluorescence.
Conclusions and discussion
(1) Different sources of TLS have different abilities to carry RNA of different fragment lengths. Further modification of TLS based on the measured data can enhance its ability to carry long RNA fragments.
(2) The self-replication function of RdRp can enhance the expression of RNA at different levels.The two different length subgenome promoters will lead to different effects of self-replication function.
(3) We performed measurements of relevant promoters and phototoxic proteins in Agrobacterium.These designs not only provide a safety guarantee for the subsequent work of plant related teams, but also facilitate the engineering operation of agrobacterium.
