Difference between revisions of "Part:BBa K3805886"

Latest revision as of 22:31, 21 October 2021

OmpT-mCherry

This composite part consists of two parts: OmpT signal peptide (BBa_K3805532) and mCherry (BBa_J06504).Outer membrane protease T (OmpT) is a 33.5 kDa endoprotease [1] located on the outer membrane of Escherichia coli.Ompt signal peptide is an excretion tag linked on the N-terminus of protein.It can transport proteins across the outer membrane with the help of OmpT. If Ompt signal peptide is fused with mCherry,it will enable mCherry to be secreted to extracellular space[2].

Usage and Biology

Designed together with modified PmrB-anti-mCherry receptor that specifically binds to mCherry (BBa_K3805238), this part acts as a two component signal transduction system whose density can be monitored by fluorescence intensity. The OmpT signal peptide in this part offers a practical and universal strategy for recombinant protein extracellular secretion in Escherichia coli.

Improvement

We fuse OmpT signal peptide at the N-terminus of mCherry to transport mCherry across outer membrane. MCherry itself cannot be secreted to the extracellular space. After the insertion of the OmpT signal peptide, mCherry is able to secrete out successfully.

Design

MCherry is a red fluorescent protein widely used in biotechnology as a tracer, including molecular labeling and localization of cell components. Due to its unique color and single molecule photostability [1], we adopted mCherry to accomplish our project. Functions of mCherry in our project are as follow: 1.Acts as reporter of the P2 promoter. MCherry is inserted at the downstream of P2 promoter, which can be activated by AIP, therefore we can determine whether P2 promoter is activated by observing the red fluorescence. 2.Acts as signal molecule that induces the cheater’s death. MCherry serves as the ligand that specifically binds to the receptor PmrB-anti-mCherry on the cheater’s cytomembrane and initiates downstream extermination pathways in cheaters, eventually leading to their death. MCherry itself, however, cannot be secreted to extracellular environment, which means that we need to modify mCherry to achieve our goals. Consequently, we insert OmpT signal peptide at the N-terminus of mCherry to help mCherry traverse the outer membrane.

Experience

We added the OmpT signal peptide sequence at the N-terminus of mCherry. To verify the function of OmpT signal peptide, we placed OmpT-mCherry on plasmid pETDuet-1 ,then transformed the recombinant plasmid into E.coli DH5α as the test group. We also constructed a recombinant pETDuet-1-mCherry plasmid without OmpT signal peptide and also transformed it into E.coli DH5α as the control group. We used fluorescent microscope and microplate reader to evaluate the transportation efficiency of OmpT signal peptide. We take pictures of the test group and the control group with fluorescent microscope. Then we use microplate reader to carry out more accurate testing experiments. According to the two groups of statistics that we collect, we carefully analyze the mCherry fluorescent intensity difference value. The difference value is calculated by the mCherry fluorescent intensity of the supernate minusing the precipitation's . The detailed description of our experiments is as follow:

Verification of OmpT signal peptide function:

1.Prepare 3 light-proof tubes of 6 mL bacteria solution respectively for the test group and the control group.

2.Observe the 6 samples with fluorescent microscope.

3.Distribute the bacteria solution in each 6 tubes evenly to 3 tubes, which means 18 tubes in total. The three tubes for each sample are named as tube A, B, and C.

4.Culture all the tubes in shaker at 180rpm, 37°C.

5.4h later, take out six A tubes from shaker and centrifugate the tubes at 8000 rpm for 10 minutes. Then separate the supernate and the precipitation. Transfer the supernate to another six light-proof tubes and add 2mL LB medium to suspend bacteria precipitation. Take 200μL from each of the 12 tubes to measure the mCherry fluorescent intensity using microplate reader.

6.Another 40 min later, take out six B tubes from shaker. Repeat step 5.

7.Another 40 min later, take out six C tubes from shaker. Repeat step 5.

Results

Figure 1 Images under fluorescence microscope.(A)mCherry without OmpT.(B)mCherry with OmpT.(Magnification:10×100).

Figure 2 The data of fluorescent density by microplate reader.The orange describes the mCherry intensity without OmpT, and the blue describes the mCherry intensity with OmpT.

With the microplate reader, we got two groups of fluorescent intensity data, each contained three samples and was measured at three points of time. If mCherry could be secreted out of cells with OmpT signal peptide, the fluorescent intensity of the bacteria solution’s supernate after supernation would be distinctly higher than the precipitation compared to the control group. Therefore, we chose the difference between the mCherry fluorescent intensity of supernate and the precipitation as the index of transportation efficiency. Here we used the method of paired sample t-test to compare the differences between the two groups of data. And the p-value we got is 1.665e-07 which was far less than 0.01. Therefore, we concluded that there were significant differences between the two groups of data, which successfully confirmed the OmpT guidance on mCherry extracellular secretion.

References

[1] Sinsinbar G, Gudlur S, Metcalf KJ, Mrksich M, Nallani M, Liedberg B. Role of Lipopolysaccharide in Protecting OmpT from Autoproteolysis during In Vitro Refolding. Biomolecules. 2020;10(6):922. Published 2020 Jun 18.Ct

[2] Sichwart S, Tozakidis IE, Teese M, Jose J. Maximized Autotransporter-Mediated Expression (MATE) for Surface Display and Secretion of Recombinant Proteins in Escherichia coli. Food Technol Biotechnol. 2015 Sep;53(3):251-260.

Sequence and Features