Part:BBa_K5154005
TEV-R5-EPG
EPG-Split TEV fusion protein with a rigid 5 amino acids linker.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 655
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 655
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 705
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 655
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 655
- 1000COMPATIBLE WITH RFC[1000]
Design
As described in the description, EPG is capable to generate action potential through calcium influx in higher animals. However, it is impossible to replicate the same mode of action in lower eukaryotes and prokaryotes. In order to overcome this issue, a separate, independent pathway needs to be constructed.
We have two options to relay the signal from EPG, either through transcription level control or through protein level control. For transcription level control, it is necessary to engineer transcription factor, allowing it to be regulated by EPG, then control the target gene expression. This setup provides flexibility, and we have proposed a design fusing EPG with LacI adapting the design from [1]. However, We discovered that the insertion site for lacI is difficult to predict, and the position of linker, type of linker will have a profound effect on the activity of protein. Considering the timescale of the project, we decide to give up this approach but switch to the protein level control.
The protein level control approach is much more straight forward, comparing to transcription level control. First, it does not require a complete redesign of the fusion protein. Most split protein reporters are designed that they will be inactivate as subunits, but will come together in specific conditions, and become activated. In our case, EPG will act as a hinge, which will effectively bring the two components together and become active. This does not require special knowledge on detailed protein engineering, instead, most split protein will be able to modified and applied on the system, with a correct selection of linkers. Second, protein level control is much more faster than transcription level control, which give the system advantage when applied to circumstances need quick response. This characteristic allows the use of feedback control which will effectively increase the accuracy of control and provide higher flexibility. Third, protein level control, especially when using protease as the payload, will allow the system being incorporated into numerous existing systems.
Figure 1 | EPG-TEV mode of action
We chose TEV as the actuator.
TEV is a protease found in Tobacco Etch Virus, has high specificity, and has been shown to retain high activity even when splitted into two parts.
We retrieved sequence of TEV, and constructed fusion protein by placing linker between N-TEV , EPG and C-TEV.
Reference
1. Liu, Meizi, et al. "OptoLacI: optogenetically engineered lactose operon repressor LacI responsive to light instead of IPTG." Nucleic Acids Research (2024): gkae479.
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