Difference between revisions of "Part:BBa K2623021"

Latest revision as of 12:01, 17 October 2018

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 insert SpyTag to its N-termini to bioconjugate with the SpyCather in the following parts. In order to test the efficiency of the OmpA-SpyTag (OmpA-ST) (see constructed parts BBa_K2623022, BBa_K2623024) transport to OMVs in our circuit, we use wtGFP, BBa_I13401 as our reporter.

Usage and Biology

As mentioned above, OmpA is constructed with SpyTag 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 the C-termini of OmpA protein (instead of the N-termini in NG1). Through the fluorescence intensity of GFP, we can compare and evaluate expression efficiency of SpyTag between NG1 and NG3. Then the better one will be uesd for downstream analysis. Here are the gene circuits of NG1/NG3 (Left Figure) and NG2/NG4 (Right Figure)

Figure 1. Diagram of NG1/3 (Left-Experiment group) and NG2/4 (Right) gene circuits (Control group)

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 on semrock[1] to find the spectrogram of wtGFP (Figure.3). So we choosed 478 nm as the maximum excitation wavelength and 512 nm as maximum emission wavelength.

Figure 3. The spectrogram of wtGFP

So we choosed 478 nm as the maximum excitation wavelength and 512 nm as maximum emission wavelength.

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 isolates 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 figure of OMVs

Figure 3. TEM figures identify our OMVs.

Building upon Yan’s lab-built high-sensitivity flow cytometer (HSFCM)[2][3], 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. HSFCM analysis of OmpA-ST-GFP inside OMVs

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.

Reference

[1] https://www.semrock.com/

[2] Zhu, SB; Ma, L; Wang, S; Chen, CX; Zhang, WQ; Yang, LL; Hang, W; Nolan, J. P.; Wu, LN; Yan, XM*. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanoparticles. ACS Nano 2014, 8, 10998-11006.

[3] Ye Tian, Ling Ma, Manfei Gong, Guoqiang Su*, Shaobin Zhu, Wenqiang Zhang, Shuo Wang, Zhibin Li, Chaoxiang Chen, Lihong Li, Lina Wu, Xiaomei Yan*, Protein Profiling and Sizing of Extracellular Vesicles from Colorectal Cancer Patients via Flow Cytometry, ACS Nano, 2018, 12, 671-680.

Sequence and Features