Part:BBa_K2623021

Bacterial outer membrane protein A (OmpA) fused with SpyTag at its N-termini and GFP at its C-termin

Summary

This part is a relatively basic part designed to target the archaeal ribosomal protein protein L7Ae and then siRNA into outer-membrane vesicles (OMVs). Porin outer-membrane protein A, OmpA, is a kind of highly expressed transmembrane porin protein of E. coli and is therefore abundant in OMVs. We inserted SpyTag to its N-termini to bioconjugate with the SpyCather in the following parts. In order to test the efficiency of anchoring the OmpA-SpyTag (OmpA-ST) (see constructed parts BBa_K2623022, BBa_K2623024) in our circuit, we use GFP, BBa_I13401 as the reporter, because the fluorescence intensity is easier to be measured.

Usage and Biology

As mentioned above, SpyTag was constructed in order to anchore L7Ae to outer membrane through the bioconjugation of the SpyTag/SpyCatcher system. We also construct another form of BBa_K2623021 (NG1)-BBa_K2623023 (NG3). The only difference is that we inserted SpyTag to C-termini of OmpA protein (instead of the N-termi in NG1). Through the fluorescence intensity of GFP, we can compare and evaluate expression efficiency of SpyTag between the this part and NG3. Then the better one would be uesd to construct our final gene circuit.

Here are the gene circuits of NG1/NG3 (Left Figure.1) and NG2/NG4 (Right Figure.2)

Diagram of NG1 and NG3 gene circuits (Experiment group)	Diagram of NG2 and NG4 gene circuits (Control group)

Figure 1. a) Genetic circuit of BBa_K2623023 (Upper pannel) and BBa_K2623021 (Lower pannel). b) Schematic illustration of these two parts. Figure 2. a) Genetic circuit of BBa_K2623024 (Upper pannel) and BBa_K2623024 (Lower pannel). b) Schematic illustration of these two parts.

Characteration

In our experiments, we cultured our bacteria transfected with these four parts respectively in 10 mL OMVs-free LB broth culture and extracted the OMVs according to our protocols, in which two forms of non-GFP OMVs were set as negative controls. Our transmission electron microscopy (TEM) picture identified what we extracted were exactly OMVs with membrane structure and diameter at about 100 nm.

TEM figure of OMVs

Figure 3. TEM figures identify our OMVs.

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 65
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1302