Part:BBa_K1378005

INPNC-linker2-MVN-linker1-mRFP

Introduction

This plasmid is a composite part used in our rTEV protease digestion assay. It uses pSB1C3 as the vector and expresses the fusion protein INPNC-MVN-mRFP. MVN is a mannan binding lectin that can specifically bind to the LPS of Microcystis aeruginosa cells (For more details please refer to the part BBa_K1378003 or our wiki). We designed a 7aa protein linker named Linker2 between INPNC and MVN which can be specifically recognized and digested by rTEV protease. Also, there is a 10aa flexible protein domain linker named Linker1 between MVN and mRFP to ensure that the fluorescence is not affected. The coding sequence is regulated by the promoter J23105, the RBS B0034 and the terminator B0015.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 466
  Illegal AgeI site found at 1915
  Illegal AgeI site found at 2027
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 1029
  

Characterization

INPNC can help us display proteins on the outer membrane of E. coli cells. However, it is still unknown whether the wanted protein is displayed outwards into the medium or inwards into the periplasm. So we designed a rTEV digestion assay to help us determine the orientation. This plasmid was used in the experimental group. To make our results persuasive, we also constructed 3 control groups: C1, C2 and C3. Plasmid C1 has the Linker2 shifted to Linker3 so that it can not be digested by rTEV protease. Plasmid C2 has the INPNC and Linker2 sequence deleted so that MVN-mRFP is not displayed on the cell surface. Plasmid C3 has the whole coding sequence from INPNC to mRFP deleted. We also included a blank group with only rTEV buffer added.

Figure 1. Gene circuits of the control groups. (a) Plamsid C1 differs to the original plasmid in that Linker2 is shifted to Linker3, which is a 8aa flexible protein domain linker that cannot be cut by rTEV protease. (b) Plasmid C2 has the coding sequence of INPNC and Linker2 deleted. (c) Plasmid C3 is a plasmid with only the promoter, RBS and terminator left on the vector.

The plasmids were all transformed into E. coli BL21(DE3) and the cells were cultured to the exponential phase. Afterwards rTEV protease as well as buffer were added to the cells harvested. The cell cultures were co-incubated with rTEV protease and centrifuged afterwards to separate the cells and MVN-mRFP that was cut down by the protease. The fluorescence intensity of the supernatant could stand for the amount of MVN-mRFP that was actually cut pff by rTEV protease and was measured by microplate reader (For experimental details please refer to our wiki). The same procedure was applied to every group for 3 times. The average results are shown in the chart below, and the fluorescence intensity data is shown in A.

Chart 1. The fluorescence intensity data measured by a microplate reader.

As can be seen from the results shown here, the fluorescence intensity of the experimental group is higher than that of the C2 and C3 groups by a large margin. Considering the precision of our equipment, this can demonstrate that the MVN-mRFP fusion protein is truly displayed outwards by using the INPNC displaying system. The results of the Experimental group and C1 group are close and it may be because of the breakage of Linker1 when we centrifuged the cells. However, the relatively high result of the C1 group is still in accordance with the demonstration.