Environmental Engineering Reference
In-Depth Information
databases with taxonomy, genome mapping, protein structure, and domain information, as
well as journal literature (http://www.ncbi.nlm.nih.gov/Genbank/index.html).
The value of complete, annotated, searchable genome sequence data for environmental
biotechnology cannot be overstated. One example is the discovery of genes analogous to a
gene of interest that may possess characteristics desirable for industrial processes or that may
be desirable in combinatorial mutagenesis (see section 2.5.3). Other examples are the location
of transposon-induced mutations or other mutations that have conferred desirable (or
undesirable) phenotypes on an organism and the elucidation of the function of a DNA
sequence that complements a mutant phenotype. Each of these contributes to the
understanding of basic biochemical processes as well as to the development of industrially-
useful bioprocesses. In addition, the progress of biotechnology is therefore significantly
enhanced by progress in genomics [4, 5].
2.3. Expression Control
Gene expression is the process by which a gene's coded information is converted into
active ribonucleic acid (RNA) and subsequently polypeptide molecules. Because the timing
and extent of expression is crucial for cellular energetic and catalytic efficiency, transcription
is controlled carefully in response to internal and external conditions. Upstream and intron-
based DNA sequences known as promoters, enhancers, and other regulatory elements, as well
as regulatory proteins known as transcription factors, are important factors in transcriptional
control. Transcriptional regulatory elements differ in strength, or the rate at which they direct
a cell to transcribe a gene into RNA, as well as in inducibility, or the stimuli to which they
respond via transcription factors [6].
2.3.1. Expression enhancement: strong and inducible promoters
. Close control of
expression of genes that direct synthesis of a desired product is advantageous and often
essential to the development of a successful bioprocess, such that the maximum amount of
substrate is converted to product while maintaining good health of the culture. One of the
earliest and still widely-utilized promoters engineered for exquisite control of heterologous
gene expression is the tac promoter, also referred to as Ptac. This sequence was designed
from a combination of the inducible lac and trp promoters in the bacterium Escherichia coli,
forming a hybrid that directs transcription up to 11 times more efficiently than the parental
sequences. This operon is repressible by the lac repressor and is inducible by addition of
isopropyl-ß-Dthiogalactoside (IPTG) [7]. Numerous other expression systems have since
been designed for both research and industrial purposes, prominently including those based
on viral T4, T7, simian virus 40 (SV40), adenovirus, baculovirus, and cytomegalovirus
promoters, the bacterial lac, araB, and xyl promoters, and yeast alcohol oxidase (AOX) and
general control (GCN) 4 promoters [8, 9].
2.3.2. Expression repression: RNA interference
. Alternatively, it is sometimes desirable
to prevent the expression of a gene altogether, or at least to diminish it significantly. While
the ideal approach to the problem of gene silencing is the deletion of the gene in question, this
is often a difficult and time-consuming process. An attractive emerging solution in eukaryotic
cells is the use of RNA interference, or RNAi, to diminish the expression of particular genes
with great specificity. This method takes advantage of an antiviral and possibly regulatory
strategy in which double-stranded RNA (dsRNA), an unusual molecule within a cell, is first
bound by a nucleolytic enzyme called Dicer. This enzyme fragments the dsRNA into
fragments of 21 to 23 nucleotides in length, known as small interfering RNAs (siRNAs),
