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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

Materials and Methods