Part:BBa_K4164020

Improvement

The improvement of NAU-CHINA this year is to integrate TEV protease and Ssr tag to construct BBa_K4164020 in order to control the degradation of mRFP and resolve the leakage problem of BBa_J04450.

TEV cleavage site (ENLYFQS) and Ssr tag (AANDENYAAV) are inserted into the C-terminus of RFP in turn (Fig.1). Meanwhile, the TEV protease is expressed also driven by Plac. 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, and RFP lost the degradation tag will no longer be degraded and begin to work.

Fig.1. Improvement of mRFP expression device.

We used pET29a+ 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 pET29a+ 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 (OD(600)) 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 OD(600) and fluorescence intensity (excitation 535 nm and emission 605 nm) per hour.

Fig.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.

Fig.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 fluorescence intensity of BBa_K4164020, BBa_J04450, BBa_K4164021, BBa_K4164022 and Control cultivated with IPTG after 12 hours;
d. the fluorescence intensity of BBa_K4164020, BBa_J04450, BBa_K4164021, BBa_K4164022 and Control cultivated without IPTG after 12 hours.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1694
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 814
  Illegal AgeI site found at 926
  
• 1000
  COMPATIBLE WITH RFC[1000]