Difference between revisions of "Part:BBa K4195101"

Revision as of 03:02, 12 October 2022

I0500-B0034-clyA-ttpB-his-B0015

Biology

ClyA

Cytolysin A (ClyA) is a pore-forming toxin that is produced by some bacteria from the Enterobacteriaceae family. When fused to the C-terminal of ClyA, heterologous proteins can be displayed on the surface of the engineered bacteria and OMVs (outer membrane vesicles) released by them (1).

TTPB

TTPB is tail tubular protein B of podophage 7. It has been found that TTPB serves as ligands that recognizes the conserved Vibrio receptor Vp0980 to mediate phage adsorption. It binds with Vp0980 of Vibrio parahaemolyticus and then mediates phage adsorption and subsequent bacterial lysis (2).

Usage and design

Engineering OMVs for treating and preventing AHPND caused by the pathogen V. parahaemolyticus are a significant part of OMEGA project (Operable Magic to Efficiently Getting over AHPND). Based on the efforts of our previous projects in 2020 (AnTea-Glyphosate) and 2021 (SALVAGE), we further developed the surface display system on the OMVs released by the engineered bacteria. The usage of cargo proteins were no more limited to enzymes that are usually utilized to catalyze series bio-chemical reactions, since some receptors or ligands involved in complex protein-protein interaction (PPI) were selected as the cargo candidates. This year, we chose two classic anchor proteins, ClyA and INPNC, to construct the display cassette with various cargo proteins including rFET (receptor), rLvAPN1 (receptor), TTPB (ligand) and TTPB (ligand) (Fig. 1). On one hand, with the receptors displayed, OMVs will gain the function of neutralizing toxins secreted by V. parahaemolyticus. On the other hand, with the assistance of ligands displayed on the surface, OMVs will become a special vector to deliver antimicrobials for the specific pathogen. In summary, we have taken a step closer to the collections of extracellular functional elements (EFE), combining the OMVs, secretion systems and surface display systems which we have been dedicated to since 2020. Learn more information from our Design page.

Fig. 1 Graphic description of the expression gene circuits for display cassette designed in OMEGA project.

TTPB-his was fused to the C-terminal of ClyA to surface display for targeting V. parahaemolyticus. Arabinose-inducible system was used in the expression circuit of this part in pSB1C3 then constructed this part. We transformed the constructed plasmid into E. coli BL21(DE3) for further verification of its expression and function on the surface of E. coli and OMVs, including the interaction between TTPB and Vp0980.

Characterization

1. Identification

When constructing this circuit, colony PCR and gene sequencing were used to verify that the transformatants were correct. Target bands (4688 bp) can be observed at the position around 5000 bp (Fig. 2).

Fig.2 DNA gel electrophoresis of the colony PCR products of BBa_K4195101_pSB1C3.

2. Characterization of displaying cargo proteins

We used BBa_I0500 promoter and RBS (BBa_B0034) to express ClyA-TTPB-his protein in E. coli BL21(DE3). The arabinose-induced overnight culture was then incubated with FITC-labeled anti-His-tag antibody in turn to verify whether the TTPB was displayed on the surface of E. coli or not.

Fig. 3 The results of immunofluorescence to characterize the function of the display system (p = 0.0019).

The ratio of fluorescence intensity (λ_Ex = 492 nm, λ_Em = 518 nm) to OD₆₀₀ of positive control (E. coli harboring BBa_K4195101) is higher than that of negative control (E. coli harboring BBa_K4195008) (Fig. 3), which indicates that our surface display system can successfully display TTPB on the surface of E. coli.

Reference

1. K. Murase, Cytolysin A (ClyA): A Bacterial Virulence Factor with Potential Applications in Nanopore Technology, Vaccine Development, and Tumor Therapy. Toxins (Basel) 14, 78 (2022).

2. M. Hu, H. Zhang, D. Gu, Y. Ma, X. Zhou, Identification of a novel bacterial receptor that binds tail tubular proteins and mediates phage infection of Vibrio parahaemolyticus. Emerg Microbes Infect 9, 855-867 (2020).

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 1205
Illegal NheI site found at 1408
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 3402
Illegal BamHI site found at 1144
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 1783
Illegal NgoMIV site found at 1795
Illegal NgoMIV site found at 2955
Illegal AgeI site found at 979
Illegal AgeI site found at 2866
Illegal AgeI site found at 3214
Illegal AgeI site found at 3280
Illegal AgeI site found at 3522
Illegal AgeI site found at 3874
Illegal AgeI site found at 4060
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI site found at 961
Illegal SapI.rc site found at 3199

@@ Line 34: / Line 34: @@
 '''Fig.2 DNA gel electrophoresis of the colony PCR products of BBa_K4195101_pSB1C3.'''
-====2. Ability of binding Vp0980 on the surface of engineered bacteria====
+====2. Characterization of displaying cargo proteins====
+We used <partinfo>BBa_I0500</partinfo> promoter and RBS (<partinfo>BBa_B0034</partinfo>) to express ClyA-TTPB-his protein in ''E. coli'' BL21(DE3). The arabinose-induced overnight culture was then incubated with FITC-labeled anti-His-tag antibody in turn to verify whether the TTPB was displayed on the surface of ''E. coli'' or not.
-We used <partinfo>BBa_I0500</partinfo> promoter and RBS (<partinfo>BBa_B0034</partinfo>) to express ClyA-TTPB protein in ''E. coli'' BL21(DE3). The arabinose-induced overnight culture was then incubated with purified Vp0980-his and FITC-labeled anti-His-tag antibody in turn to verify whether the displayed TTPB could bind Vp0980 or not.
+[[File:T--XMU-China--CtB.png|300px]]
-====3. Ability of binding Vp0980 on the surface of OMVs====
+'''Fig. 3 The results of immunofluorescence to characterize the function of the display system (p = 0.0019).'''
-For the test on OMVs, the OMVs were firstly extracted from the culture of engineered bacteria harboring <partinfo>BBa_K4195121</partinfo> after induction. Subsequently, the OMVs-containing samples were directly spotted onto the nitrocellulose (NC) membrane. Then the NC membrane was incubated with purified Vp0980-his and anti-His-tag antibody in turn and finally probed by the HRP-conjugated secondary antibody(''3, 4''). By comparing the chemiluminescence imaging results of OMVs-containing samples of different origins, we could characterize whether the displayed TTPB on OMVs is functional or not.
+The ratio of fluorescence intensity (''λ''<sub>Ex</sub> = 492 nm, ''λ''<sub>Em</sub> = 518 nm) to OD<sub>600</sub> of positive control (''E. coli'' harboring <partinfo>BBa_K4195101</partinfo>) is higher than that of negative control (''E. coli'' harboring <partinfo>BBa_K4195008</partinfo>) (Fig. 3), which indicates that our surface display system can successfully display TTPB on the surface of ''E. coli''.
 ===Reference===
@@ Line 49: / Line 49: @@
 . M. Hu, H. Zhang, D. Gu, Y. Ma, X. Zhou, Identification of a novel bacterial receptor that binds tail tubular proteins and mediates phage infection of Vibrio parahaemolyticus. ''Emerg Microbes Infect'' '''9''', 855-867 (2020).
-. J. L. Valentine ''et al''., Immunization with Outer Membrane Vesicles Displaying Designer Glycotopes Yields Class-Switched, Glycan-Specific Antibodies. ''Cell Chem Biol'' '''23''', 655-665 (2016).
-. T. C. Stevenson ''et al''., Immunization with outer membrane vesicles displaying conserved surface polysaccharide antigen elicits broadly antimicrobial antibodies. ''Proc Natl Acad Sci U S A'' '''115''', E3106-E3115 (2018).