Team:Greensboro-Austin/MAPs

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The mussel

The mussel is a marine mollusk (most commonly known for its tastiness) that lives in the intertidal zone on wave washed rocks. To stand the tidal forces, they must bind to these rocks with their endogenous glue. This glue's properties make it an attractive target for synthetic improvement.

Mussels have an organ called the foot, which is used to both pull the mussel through terrain and to excrete a structure called the byssus. These byssal threads are made up of collagen and protein and are what allows the mussel to attach to surfaces. There are several different proteins that make up the byssal threads, but the ones we’re most interested in are the ones that lie at the bottom of the structure. These proteins are responsible for the actual binding to the substrates and therefore have the most potential for use as a glue.

So why are mussel adhesion proteins (MAPs) more useful over traditional glues?

1. They can attach to many different types of surfaces, including both organic and inorganic ones.
2. They are strong and durable, able to withstand the harsh ocean environment.
3. They’re made of proteins so they can be easily degraded and are environmentally friendly.
4. Unlike most glues, they can work and be cured underwater.
5. They are biocompatible (nonimmunogenic).

Project

(insert abstract here)

Make mussel adhesive proteins (MAPs) using E. coli Incorporate unnatural amino acids (How? Expand in Introduction. Stress novelty of idea.)

Natural vs. commercial underwater adhesives (Table of properties? Strengths, biocompatibility, cost, availability)

Benefits of synthetic MAPs (Why they're worth engineering)

Introduction

explain different surgical glues, downfalls of current natural ones, and emphasize potential of MAPS

Recombinant mussel adhesive proteins from Mytilus galloprovincialis

Explain properties of fp-1,2,3,4,5, dopamine, tyrosine, L-dopa, dopaquinone

explain fp 151 = fp 1 + 5 + 1

1 is a 6x repeat of 10 aa sequence

Study by (CITE SOURCE) showed that fp-151 was stickiest(?)

L-dopa can be produced through post-translational oxidation of tyrosines

---inefficient due to requirement of tyrosines from outside

Explain what RF1 does, and what suppressing it will do

-what is RF0, what strain (CITE SOURCE, give credit to lab)

-substitute ambers with some other stop codons so they still stop in RF0 strain

synthesize L-DOPA with UAAs to improve efficiency of production, and have more control on synthesis

-cell must incorporate L-dopa in tRNA/synthetase (name of plasmid, CITE SOURCE, credit the lab giving us plasmid)

Thus, AMBER codons are read as L-DOPA instead of STOP

PRIMARY GOAL: make MAPs with increased levels of L-DOPA, produced through in vivo UAA incorporation

future:

test different numbers of L-DOPAs in each of 3 geneblocks

express MAPs on cell surface, using Berkeley '09 mechanisms to get sticky microbes

or use purification techniques to extract product from the microbes

measure stickiness directly with materials techniques

measure stickiness in comparison with normal e coli (what % stick to glass in culture?)

directed evolution to obtain improved stickiness

-grow surface MAP-making e coli in flasks

---make sure e coli have the resources to mutate/incorporate L-DOPA normally

-wash the ones that didn't stick

-keep growing

-examine MAP structure after several generations

Strategy

Unnatural Amino Acid Incorporation

A tyrosyl-tRNA synthetase from Thermus thermophilus (PDB: 1H3E)

Previous studies have generated L-DOPA residues through post-translational modification (Choi et al. 2012, Hwang et al. 2007). However, this method has the disadvantage of less control of where L-DOPAs are incorporated and how efficient the process is. We will directly incorporate L-DOPA as an unnatural amino acid (UAA) into the MAP.

Successful UAA incorporation requires the addition of two components: a new tRNA synthetase and its corresponding tRNA. This pair must be orthogonal, meaning that they must be highly specific for each other and the UAA. The tRNA should also be complementary to the UAG stop codon (also known as the “amber” stop codon), which will be reprogrammed to code for the new UAA instead of termination.

Schultz et al. have developed a system for UAA incorporation. For a starting point, they obtained tyrosyl-tRNA and tyrosyl-tRNA synthetase from Methanococcus jannaschii.This pair was chosen since the tRNA synthetase doesn’t bind to the codon binding region on the tRNA, allowing it to be reprogrammed to bind to the amber stop codon. Through a series of positive and negative selection cycles on both the tRNA and the tRNA synthetase, they were able to create a functional UAA incorporation system. We will be employing Schultz’s L-DOPA pair as shown in the circuit diagram below.

However, this pair is not perfect, since it is not completely orthogonal and will sometimes incorporate tyrosine instead of L-DOPA. For future work, it is possible to evolve a better system that is more specific.