Team:NCTU Formosa/Parts
Overview
The main goal of our project, E. Hybrid, was to develop a brand-new strain of E. coli that
could enhance protein expression by using light as energy source. We expected that it paves a
promising avenues for bioengineering usages.
We summited Harmonized Gloeobacter rhodopsin(GR) with GFP linker as basic part
and also its composite part with T7 promoter and RBS. We also used BBa_K274200 & BBa_J04450 in our
project. All BioBricks applied in our project are
sequence-confirmed and the following table shows our new parts:
<groupparts>iGEM20 NCTU_Formosa</groupparts>
Harmonized Gloeobacter rhodopsin (GR) with linker and GFP
Gloeobacter rhodopsin introduction
GR is a light-driven proton pump that originates from the primitive cyanobacteria,
Gloeobacter violaceus. It is a seven helix membrane protein located in the inner membrane.
Acting as a light-driven proton pump, GR can transfer
protons from the cytoplasmic region to the periplasmic region following light absorption. That is,
it establishes the proton motive force to push ATP synthase transforming solar energy into universal
energy currency, ATP. The reason
that GR has a function with light is its specific chromophore, all-trans-retinal. It changes its
conformation when induced by light, resulting in a series of protonated and deprotonated reactions
on the several amino acids in GR and
causing the transfer of protons.
Modifications of GR for better folding & expression
Harmonized GR is different from the common GR. It's been treated under harmonization,
one kind of codon optimization. Since the codon frequency of GR in wild-strain and our host-strain
is different, we use harmonization, which is an algorithm,
to optimize our sequence of codons but without changing the sequence of amino acids.
GFP linker vs. Correct Protein Folding
The linker is Gly and Ser rich flexible linker, GSAGSAAGSGEF, which provides performance
same as (GGGGS) 4 linker, but it does not have high homologous repeats in DNA coding sequence.
Therefore, if GFP expresses well, we can ensure that
GR proteins fold robustly and are fully soluble and functional. Furthermore, flexible linker could
keep a distance between functional domains, so GFP would not interfere the function of GR.
T7 promoter + LacO + RBS + Harmonized GR with linker and GFP + 6x His-tag + Terminator
This composite part is regulated by a T7 promoter. A polyhistidine-tag is used for
purification. In order to assess the expression of GR, we used a linker sequence in conjunction with
GFP.