Overview
This year, our team submitted some documents from experiments to several different parts.
Characterize Existing Parts
We completed the experimental characterization of the parts (BBa_K3378000, BBa_K185047, BBa_K185047, BBa_K3733011,BBa_K1231000) and added some information about them.
ClyR
This chimeric lysin can bind to S. mutans with a cell-wall binding domain and then lyse its cell wall from outside by a catalytic domain. ClyR has no activity for Gram-negative bacteria and can be expressed in Escherichia coli.With the guidance of appropriate signal peptide, ClyR can be secreted to the outside of the cells, killing S. mutans in the environment. We have successfully validated ClyR demonstrates a certain level of bactericidal activity, which becomes increasingly significant as its concentration increases. The experimental results are shown in the figure below.
Figure 1. Variation curve of OD600 of S. mutans UA159 treated with different ClyR concentrations.
For more information you can see here: BBa_K3378000
LuxR-Lux pR-sfGFP
This composite part is used to detect the response of LuxR (BBa_C0062) to AHL signaling molecules. When LuxR receives the signal of AHL, it can regulate the downstream Lux pR (BBa_R0062) to make superfolder GFP (BBa_K4115000) express.This part is togther used with J23101-B0034-LuxI-B0015 (BBa_K4115040).
We have successfully validated that E.coli containing this part would show stronger fluorescence when cultivated with E.coli containing BBa_K4115040 than with blank E.coli, which would not show distince fluorescence.
Figure 2. Fluorescence intensity in different value of OD600.
For more information you can see here: BBa_K4115039
ReIE
As a toxin protein, it does not have strong bactericidal effects, but has strong abilities to inhibit bacterial growth. We can use this toxin protein to reduce the influence of environmental temperature fluctuations on expression leakage of the RNA thermosensor. It can also play a role in inhibiting engineered bacterial growth in the early stage before the temperature-sensitive circuit activates engineered bacteria suicide. Its bacteriostatic effects are rapid and work well in the early stage according to our experiment.
Figure 3. Bacterial OD600 over time.
Figure 4. Viable cell counts over time.
For more information you can see here: BBa_K185047
HepT
HepT is a toxin, a member of the higher eukaryotes and prokaryotes nucleotide-binding (HEPN) superfamily, strongly inhibiting cell growth in S.oneidensis and Escherichia coli. The HepT toxin (HEPN-domain protein) could function as an RNase with a RX4-6H active motif and cleave mRNA to inhibit cell growth.
Figure 5.Bacterial OD600 over time.
For more information you can see here: BBa_K3733010
RNA thermometer
The RNA thermometer has a high fold change between 26°C and 36°C. We used two methods to experimentally verify it. We also used DINAMelt's server for analysis of the stem-loop structure.
Figure 6. Analysis of Differential Relative Fluorescence Intensity under Four Hours of Different Temperature Induction Conditions.
Figure 7. Bacterial OD600 over time.
Figure 8. Phenotypic result comparison chart.
For more information you can see here: BBa_K3733011
Pasr
This is a promoter that is activated in an acidic environment. It is the promoter of the acid shock protein ASP in E. coli. promoter of the acid shock protein ASP in E.coli often functions under acidic stress conditions. Since its sequence contains a series of binding sites for regulatory proteins such as the RstA box, its activity can be well artificially regulated and altered. Therefore, it can be well utilized in synthetic biology circuit design. Some experimental teams have combined pH sensing with toxin expression to create an effective bacterial containment system. We believe the functionality of this promoter in responding to environmental pH changes could provide some reference meaning to projects detecting changes in environmental pH conditions.
Figure 9. Relative fluorescence intensity over time under different pH induction.
Figure 10. Differential analysis of relative fluorescence intensity after 4 hours of induction under different pH.
For more information you can see here: BBa_K1231000