Biology Reference
In-Depth Information
Partially due to a lack of facile gene writing tools, groups in synthetic biology took on a
standardized assembly approach to construct gene circuits based on the collection and
standardization of biological parts termed BioBricks. The BioBrick library is an open source
standardized collection of biological parts, which is growing in size and documentation.
BioBrick
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or BglBrick
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methods offer standardization in assembly. Standardized flanking
sequences containing restriction sites allow for pairwise assembly of modular constructs into
larger constructs. This makes assembly more parallel than traditional restriction digest and
ligation methods. Efforts have been made to refine and automate the process.
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However,
larger constructs are often incompatible with BioBrick assembly due to additional restriction
sites within the construct. Also, BioBrick digest scars cannot be avoided. The sequential
BioBrick assembly procedure also makes it laborious.
While we now have the ability to sequence entire genomes and are able to computationally
simulate dynamic systems to a certain extent, the ability to rapidly implement designed
circuits that perform as expected still eludes us. Gene synthesis can serve to replace
laborious recombinant DNA methods used in traditional genetic engineering. It can serve to
replace BioBrick assembly, or at least circumvent the need to assemble smaller constructs.
It would enable the transition from ad hoc restriction enzyme methods to systematic gene
circuit and pathway designs.
Gene synthesis would ease the transition of synthetic biology from modules to systems.
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This intellectual investment in DNA synthesis may enable scientists and engineers to quickly
design, construct, and validate gene circuits that may do anything from curing diseases to
generating alternative energy in a synthetic fashion.
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Codon Optimization
The demand for efficient recombinant protein production has grown in recent years.
Commercially valuable proteins can be expressed in heterologous hosts like
E. coli
for
large-scale production. These hosts can be used to produce a wide array of useful proteins
for applications like pharmaceutics, cell therapies, and nano-material production. Even
though the chemical and industrial setups have been engineered and optimized, biological
protein expression can remain a bottleneck in production systems.
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It is acknowledged that modifying a synthetic gene design for optimal synonymous
codon usage using DNA synthesis can help achieve increased protein expression levels.
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Unfortunately, robust rules for codon optimization are not available beyond the notion
of mimicry of naturally occurring highly expressed genes. There is a lack of experimentally
supported design principles based on codon usage, because of the limitation of small
sample size for study imposed by existing synthesis technologies. Current methods of codon
optimization are slow and expensive, due to the lack of fast and economical gene synthesis
technology and incomplete understanding of protein translation.
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Translation in bacteria
involves initiation, elongation, termination, and ribosome turnover. At the same time,
interactions with the expression environment vary protein solubility, mRNA stability,
protein localization, and cell viability. Most factors are not fully understood, making it
difficult to postulate causality. A number of theories have been proposed in the past for
codon optimization. Some of the traditional schemes include CAI (codon adaptation index)
maximization, codon sampling, dicodon optimization, and codon frequency matching.
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Other theories for codon optimization include the importance of number of rare
codons, pairs of consecutive rare codons, frequency of rare-pairs, number of potential
RNaseE cleavage sites, Shine-Dalgarno occlusion by secondary structure, number of
predicted rho-independent transcription termination signals, and many others.
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Kudla et al.
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performed a systematic study where they generated a library of 154 GFP
codon variants in which the codon usage was varied randomly at synonymous sites.
