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) and NG2/NG4 (Right Figure)
Figure 1.left a) Genetic circuit of BBa_K2623021 (Upper pannel) and BBa_K2623023 (Lower pannel). b) Schematic illustration of these two parts. Figure 1.right a) Genetic circuit of BBa_K2623022 (Upper pannel) and BBa_K2623024 (Lower pannel). b) Schematic illustration of these two parts.
We search https://www.semrock.com/to find the spectrogram of wtGFP (Figure.3)
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(Figure 3).
Figure 3. TEM figures identify our OMVs.
Building upon Yan’s lab-built high-sensitivity flow cytometer (HSFCM, link to http://xmyan.xmu.edu.cn/), we successfully detected the OMVs labelled with GFP and evaluated the efficiency of its transport to OMVs (Figure 4). Herein, we'd like to extend our heartfelt thanks toward Yan's lab due to the very kind and selfless help during the experiment.
Figure 4 show bivariate dot-plots of GFP green fluorescence versus side scattering (SSC) for our OMVs isolates. E. coli transfected with BBa_K2623021 (Figure 4a) could secret more OMVs labeled with GFP than E. coli transfected with BBa_K2623022 (Figure 4b). Similarly, E. coli transfected with BBa_K2623023 (Figure 4c) would secret more OMVs labeled with GFP than E. coli transfected with BBa_K2623024 (Figure 4d). Moreover, we could see E. coli transfected with BBa_K2623021 (Figure 4a) would transport OmpA-ST to OMVs more efficiently than E. coli transfected with BBa_K2623023 (Figure 4c). The remarkable difference of GFP-OMVs ratio between these two parts might account for the different inserted site of ST. SpyTag (ST) in BBa_K2623021 (Figure 4a) is inserted to N-termini of OmpA, while ST in BBa_K2623023 (Figure 4c) is inserted to C-termini of OmpA. Our HSFCM data shows that BBa_K2623021 is more efficient to transport ST-OmpA-GFP to OMVs and might be a good candidate for the following study.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 65
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1302
| emission | 510 nm |
| excitation | 478 nm |