Background

The Ssr tag exists in bacteria extensively, serveing as a central feature of protein-quality control system. When protein translation stalls, the ribosome-rescue system mediates the addition of the sequence AANDENYALAA, an Ssr tag, to the C-terminus of the nascent polypeptide. The peptide sequence will lead the modified protein to degradation. In addition, Tobacco Etch Virus (TEV) protease is a site-specific protease which has the ability to recognize the amino acid sequence consisting of ENLYFQS and cleave it between Q and S. Taking advantage of the characteristics of TEV protease and Ssr tag, we can integrate a logic operation to decrease the leakiness of parts.

P_lac promoter is a widely used promoter in induced expression of proteins. The gene products of lacI is a repressor for lac operon which can bind to lac operator, then block the RNA polymerase from binding to the P_lac promoter and switching on transcription. When induced by IPTG, LacI can be removed from the site so that the transcription of downstream gene will be turned on. BBa_J04450 is a Red Florescence Protein (RFP) expression device driven by P_lac promoter. When it works in E. coli , a quantity of RFP will express without IPTG due to the low expression level of LacI in the chassis. JNFLS-2020 has managed to reduce the leakiness by linking lacI and RFP to the downstream of P_lac. This year, NAU-CHINA tried to build an anti-leakage device on BBa_J04450 which can be stretched to extensive inducible promoter.

The improvement of NAU-CHINA this year is to resolve the leakage problem of BBa_J04450. In order to control the degradation of mRFP, we integrated TEV protease and Ssr tag to construct the part BBa_K4164020. TEV cleavage site (ENLYFQS) and Ssr tag (AANDENYAAV) are inserted into the C-terminus of RFP in turn (Fig.1). Meanwhile, the expression of TEV protease is also driven by P_lac. The Ssr tag can lead to the degradation of RFP expressed without IPTG. When induced by IPTG, the TEV protease can be inductively expressed and cleave specifically at the cleavage site, which results in the loss of Ssr tag in RFP. The RFP, which lost the degradation tag, will no longer be degraded and begin to work.

Figure 1. Improvement of mRFP expression device

We used pET-29a(+) as the vector, which is capable of expressing LacI to conduct BBa_K4164020. Additionnaly, we constructed BBa_J04450, BBa_K4164021 and BBa_K4164022 (Fig.2) in pET-29a(+) as control groups. These plasmids were transformed into E. coli BL21 respectively and grown in LB medium at 37℃ until the optical density at 600nm (OD600) reached 0.6. After that, the bacteria culture fluid was incubated in 50mL LB medium with or without IPTG induction at 37℃ for 12 hours, and measured the OD600 and fluorescence intensity (excitation 535 nm and emission 605 nm) per hour.

Figure 2. a.Improved part BBa_K4164020; b. Original part BBa_J04450; c. Control part 1 BBa_K4164021; d. Control part 2 BBa_K4164022

We find that the leakage level of BBa_K4164020 is lower compared with BBa_J04450 and BBa_K4164021. What’s more, due to the absence of TEV protease, the fluorescence intensity of BBa_K4164022 is poor regardless of whether there is IPTG induction. However, the relative fluorescence intensity of BBa_K4164020 and BBa_J04450 are similar in the condition of IPTG induction, which suggests the Ssr tag has little influence on the expression of RFP under normal circumstances.

Figure 3. The result of improvement. a. the variation of fluorescence intensity/OD of BBa_K4164020, BBa_J04450, BBa_K4164021 and BBa_K4164022 cultivated with or without IPTG induction; b. the fluorescence intensity/OD of BBa_K4164020, BBa_J04450 and BBa_K4164021 cultivated without IPTG induction; c. the phenotypic photos of BBa_K4164020, BBa_J04450, BBa_K4164021, BBa_K4164022 and Control cultivated with IPTG after 12 hours; d. the phenotypic photos of BBa_K4164020, BBa_J04450, BBa_K4164021, BBa_K4164022 and Control cultivated without IPTG after 12 hours.

Zhang, Yan, et al. "Design and optimization of E. coli artificial genetic circuits for detection of explosive composition 2, 4-dinitrotoluene." Biosensors and Bioelectronics 207 (2022): 114205.

Farrell, Christopher M., Alan D. Grossman, and Robert T. Sauer. "Cytoplasmic degradation of ssrA-tagged proteins." Molecular microbiology 57.6 (2005): 1750-1761.

Taxis, Christof, and Michael Knop. "TIPI: TEV protease-mediated induction of protein instability." Ubiquitin family modifiers and the proteasome Humana Press, 2012. 611-626.