Difference between revisions of "Part:BBa K2623030"

Latest revision as of 18:58, 17 October 2018

A composite part of OmpA-SpyTag and SpyCatcher-L7Ae-mRFP1

Summary

This part is a composite part constructed by linking BBa_K2623024 and BBa_K2623025 together. BBa_K2623024 can express OmpA inserted with SpyTag (ST) at its C-termini. BBa_K2623025 can express archaeal ribosomal protein protein L7Ae inserted with SpyCatcher (SC) at its N-termini and mRFP1 at its C-termini under the induction of IPTG at 0.5 mM concentration. It's another form of BBa_K2623028.

Usage and Biology

Though we have revealed the OmpA-ST inside OMVs (BBa_K2623021), we are inclined to figure out the ST/SC bioconjugation inside OMVs. Once IPTG is added, L7Ae could be expressed with SpyCatcher and RFP modification and then be encapsulated into OMVs through ST/SC conjugation. We also construct another form of BBa_K2623030 (NG9-1)-BBa_K2623028 (NG8-1).The only difference is that the latter part links BBa_K2623022 (NG2) with NG5 instead of NG4 (Figure 1). Through the fluorescence intensity of mRFP1, we can compare and evaluate the efficiency of SpyTag/SpyCather bioconjugation inside OMVs . Then the better one would be uesd to construct our final gene circuit-BBa_K2623032.

Figure 1. a) Genetic circuit of SC-L7Ae-RFP. b) The spectrogram of mRFP1[1].

Characterization

In our experiments, we cultured our bacteria transfected with these four parts (BBa_K2623028, BBa_K2623029, BBa_K2623030, BBa_K2623031) respectively in 100 mL OMVs-free LB broth culture and extracted the OMVs according to our protocols, in which two forms of non-RFP OMVs were set as negative controls. By using Yan’s lab-built high-sensitivity flow cytometer (HSFCM)[2][3], we successfully detected the OMVs labelled with RFP and evaluated the efficiency of the ST/SC bioconjugation inside OMVs (Figure 2). 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 2. HSFCM analysis of ST/SC bioconjugation inside OMVs	Figure 3. HSFCM analysis of OmpA-ST-GFP inside OMVs

Figure 2 contains bivariate dot-plots of RFP red fluorescence versus side scattering (SSC) for our OMVs isolates. E. coli BL21 transfected with BBa_K2623028, BBa_K2623029, BBa_K2623030, BBa_K2623031 were incubated with OMVs-free LB culture for about 5 hours to get a 0.6-0.8 OD600 respectively, in which BBa_K2623029 and BBa_K2623031 were set as negative controls. Then IPTG was added to a final concentration at 0.5 mM and nurture the bacteria overnight. OMVs were isolated according to our protocols and then analyzed by HSFCM. It’s interesting to note that E. coli transfected with BBa_K2623030 (Figure 2c) could secret more RFP-OMVs than BBa_K2623028 (Figure 2a), indicating that ST/SC conjugation inside OMVs is more efficient with ST at the C-termini of OmpA.

This result is inconsistent with the result shown in BBa_K2623021 (Figure 3), in which ST at the N-termini of OmpA (BBa_K2623028) has a higher efficiency to be encapsulated inside OMVs. We propose that GFP inserted to OmpA might interfere the transport of OmpA-ST (BBa_K2623021 and BBa_K2623023) to OMVs.

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