S.tar, by iGEM Technion 2016

Intein - Introduction

Introduction

In our attempts to expand our system and design more receptors, we realized that our original approach of creating chimeric receptors will most likely work only by fusing the Tar chemoreceptor with other Ligand Binding Domains (LBD) from the bacterial world.

We decided to attempt a new approach that was proven in LIANG et al (3). According to it, it would be possible to take receptors from other organisms and create a switching mechanism by using an intein protein. As a proof of concept we tried the human estrogen receptor - hERɑ that binds estrogenic compounds.

Inteins

An intein is a protein element found as an in-frame insertion within the sequence of a particular host gene. It possesses the ability to excise itself post-translationally from its protein host, and ligate the flanking peptide sequences with a native peptide bond via a very efficient self-catalyzed reaction, called protein splicing.

Fig.1: Inteins, valuable genetic elements in molecular biology and biotechnology.

Conditional protein splicing

Splicing is the name of the process in which an intein excises itself out of the flanking protein segments. This process can be conditioned to be activated by an external trigger such as a small molecule, light or even temperature. (7)
We attempted to engineer a molecule controlled intein. Such a construct was successfully designed in the past by fusing a ligand binding domain into the intein (7). This domain undergoes a conformational change upon ligand binding and induces splicing by doing so. Using this method we can remain compatible with S.Tar's overall approach of changing ligand binding domains. By changing the LBD which is fused into the intein, it would become sensitive to different substances.

Fig.2: Recent advances in in vivo applications of intein-mediated protein splicing.

Design and Implementation

Detection using chemotaxis and inteins:

One of the LBDs used with inteins is that of hERɑ - a human estrogen receptor, which was inserted into an intein and fused to lacZ as a proof of concept at (2,6).
We tried the same construct, the only difference being that we fused the intein into the Tar receptor in critical locations, which will render Tar inactive - “off mode”.

Upon contact with an estrogenic compound - the hERɑ-LBD will bind to it, and undergo a conformational change, which will induce intein splicing. During the process the intein will ligate the Tar back to it’s native form and enable chemotaxis - “On mode”.

Figure 3:
a. Plasmid construct containing the intain gBlock in the Tar chemoreceptor.
b. General form of Tar chemoreceptor with and without the intein.
c. The overall switching strategy.

Our intein switch

The Saccharomyces cerevisiae VMA intein has been shown to retain its activities after being transferred into non native genes or cell hosts.
The VMA intein contains two regions:
1. Splicing region.
2. Endonuclease region.
The latter one is not essential for protein splicing.

Initially, we found a Biobrick of an intein with hERɑ-LBD designed by Carnegie Mellon iGEM2014 team.
However, this design was not successful in their experiments, thus we designed an original gBlock using the VMA intein in two steps:
1. The endonuclease region of the VMA intein (which is not essential for splicing) was replaced with a cDNA coding for the LBD of the human estrogen receptor (8).
2. Four restriction sites (EcoRI, XbaI, SptI, PstI) were deleted by silent mutations (These enzymes exist in iGEM plasmid backbones pSB1C3).

Fig.4: Basic structure of our designed gBlock with the hERɑ ligand binding domain, instead of the original endonuclease region of the intein.

Procedure

Two separate strains were generated by inserting the intein-gBlock in two different locations in the Tar receptor, following the requirements for efficient protein splicing:
- The first amino acid of the C-terminal extein should be a cysteine (Cys), serine (Ser), or threonine (Thr).
- The last amino acid of the N-terminal extein should be alanine (Ala) (1,3).

Fig.5: Demonstration of insertion of intein gBlock into Tar chemoreceptor, with the requirement for efficient protein splicing.

The aim was to insert the intein into the LBD region, rather than the cytoplasmic region, due to several reasons:
1. Prevent interruption of the signal peptide (the sequence responsible for signaling the cell to migrate the protein to the membrane), that is assumed to be in the C-terminus of the protein.
2. Avoid interruption of the transmembrane region.

Two regions in Tar-LBD have been found to answer the requirements, and for each we modeled the structure of Tar-intein complex, using Phyre2 Fold Recognition server (4):

Fig.6: a. Location 1 - between Ala101 and Cys102. b. Location 2 - between Ala118 and Cys119. c. Native Tar receptor

Challenges

This approach has never been done in any research, so we were heading into unknown territory. There were a few major questions that needed to be answered:
1. Will Tar-intein chimera reach its proper location in the cell membrane?
2. What happens if the conformational change is happening while the chemoreceptor is in the membrane?
3. Will the intein splice out when it binds estrogen?
4. Will Tar fold correctly after intein splicing?

Designed experiments

Several experiments are required to answer said questions.

1) In order to verify that the Tar-intein chimera migrated to the proper location in the cell membrane a GFP will fused to the C-terminus of Tar. This will enable tracking of the protein in the cell by using fluorescence microscopy.
2)To validate our switching mechanism - the new E.coli strains will be incubated with 17-beta-estradiol for 2 hours to trigger intein splicing. Afterwards a chemotaxis assay will be performed with Aspartate (the native Tar ligand) as an attractant, to check whether the strain has an active Tar, and is able to move.

Results

We were unable to insert the intein-gBlock in the Tar receptor via Gibson assembly. Only a short part of the intein was succefully assembled. We believed the intein to be too long (1575 bp) which is why new intein-gBlocks were generated, by splitting the original-intein-gBlock into two parts. An attempt to insert these parts into Tar by Gibson assembly has failed as well. In our third and final attempt we used a different cloning method - blunt ligation. In this attempt, the original-intein-gBlock (one part) was used. This method unfortunatly did not work as well.

Outlook

Long fragment cloning is often challenging due to the fact that cloning efficiency is reduced with fragment size. Using optimized cloning products might be essential.
As cloning methods did not work well, the next stage is to assemble the two-parts intein gBlocks via Gibson assembly, in order to test whether it can be assembled to form a single intein segment. Following that, cloning of the segment into Tar using blunt ligation.
We believe this novel approach has the potential to produce new E.coli strains which contain a Tar-switch.
Except for material detection this approach can also be used as a biological AND gate - where we could verify if two specific materials are found in a sample.

Referances

1. CHONG, Shaorong; XU, Ming-Qun. Protein splicing of the Saccharomyces cerevisiae VMA intein without the endonuclease motifs. Journal of Biological Chemistry, 1997, 272.25: 15587-15590.‏

2. ELLEUCHE, Skander; PÖGGELER, Stefanie. Inteins, valuable genetic elements in molecular biology and biotechnology. Applied Microbiology and Biotechnology, 2010, 87.2: 479-489.‏

3. LIANG, Rubing; ZHOU, Jing; LIU, Jianhua. Construction of a bacterial assay for estrogen detection based on an estrogen-sensitive intein. Applied and environmental microbiology, 2011, 77.7: 2488-2495.‏

4. Phyre2 modeling server online.

5. SHIOMI, Daisuke, et al. Helical distribution of the bacterial chemoreceptor via colocalization with the Sec protein translocation machinery. Molecular microbiology, 2006, 60.4: 894-906.‏

6. SKRETAS, Georgios; WOOD, David W. Regulation of protein activity with small‐molecule‐controlled inteins. Protein Science, 2005, 14.2: 523-532.‏

7. TOPILINA, Natalya I.; MILLS, Kenneth V. Recent advances in in vivo applications of intein-mediated protein splicing. Mobile Dna, 2014, 5.1: 1.‏

8. Uniprot‏