Biology Reference
In-Depth Information
downstream DNA sequences of the target DNA, so that the complementary overhangs
will anneal to each other and be extended during the PCR process, which allows ordered
assembly. Circular Polymerase Extension Cloning (CPEC) was further developed and
enables the assembly of a target gene into a linearized plasmid in PCR implementation.
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These PCR-based technologies avoid restriction digestion and ligation, though their
application is limited by high GC content sequences. Scale-up is problematic due to the
error rate of PCR, especially in construction of DNA molecules with sizes over 10 kb.
Similar to the overlap extension strategy, but not PCR-based, the uracil specific excision
reagent (USER) method can also be used for large pathway construction.
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One uracil
nucleotide is recruited in DNA fragments, close to the 5
0
-end of the linear fragment,
and the fragments are excised by uracil DNA glycosylase along with cleavage by
apurinic/apyrimidinic (AP) endonuclease. This produces complementary overhangs
which can be fused together by T4 DNA ligase which makes the desired product ready for
PCR amplification. Sequential implementation enables the construction of an entire
pathway in plasmid, or even in the chromosome.
Sequence and Ligation Independent Cloning (SLIC) utilizes in vitro homologous
recombination and single-strand annealing to assemble multiple fragments in a single
reaction.
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In both the inserted fragments and vector, single-stranded DNA overhangs
are produced by the chew-back mechanism of exonucleases. T4 DNA polymerase bears
some exonuclease activity, but this reaction can be then stopped by the addition of dNTP.
Thus, this enzyme was chosen to chew-back the DNA. Assembly of 10 fragments with sizes
ranging from 275 bp to 980 bp into one plasmid has been proven with the SLIC method.
PATHWAY OPTIMIZATION
Strategies for Optimizing a Metabolic Pathway Based
on Gene Expression
The methods which allow facile assembly of a full pathway have opened many doors for
biosynthetic processes. However, it is not as simple as stringing multiple genes together
because the pathway must be optimized to overcome the metabolic burden. Initial efforts to
balance the gene expression in a pathway to minimize the metabolic burden were focused
on varying inducer concentrations. It has been shown that different inducer concentrations
can greatly affect the titer of the desired product.
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However, more advanced tools have
since been developed to diversify the available toolkit.
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COPY NUMBER OF PLASMIDS EXPRESSING A TARGET PATHWAY
During initial work in this field, a high-copy-number plasmid, which replicates 30
40 times
within the cell, was thought to be the vector of choice when biosynthetically producing
value-added compounds because it produced a larger amount of proteins compared to
low-copy-number plasmids. However, Jones et al.
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proved that low-copy-number plasmids,
which replicate only 1
2 times within the cell, could actually produce a higher yield of
the product. Two independent pathways were analyzed: the production of polyphosphate
(polyP) and lycopene. These pathways were chosen for the study because the substrates
required to make the metabolites were critical for general cell metabolism, and each
metabolite could be produced/manipulated by the overexpression of a single gene.
The metabolic burden was quantified based on the final optical density (OD) reading
of the cultures containing each of the two independent metabolic pathways. It was shown
that the single copy plasmids overexpressing the key enzyme for both pathways had a higher
OD than the culture with the analogous high-copy plasmids. This higher OD increased
the overall yield of the desired products in the low-copy plasmid system. Thus, more cells
producing a smaller amount of compound can be more productive than fewer cells
producing a lot of the compound. This method of lowering the copy number of the plasmid
