Gene Design
Designing genes de novo liberates scientists from the constraints of naturally occurring nucleotide sequences. However, the enormous number of base pair combinations that all code for the same amino acid sequence can be daunting. On average, each amino acid is represented by three different codons. For a small protein of only 200 amino acids, then, there are around 3200 (or ~2.5*1094) nucleotide sequences to choose from. Eurogentec's original gene design software focuses on the need for codon concentrations that results in high levels of heterologous protein expression. Our proprietary design algorithm balances a host of relevant factors to produce the most effective gene sequence possible.
1. Selecting a codon usage table: The large number of genomic sequences now available has made it possible to derive the codon usage for any organism. Eurogentec can design genes based on the codon usage tendencies of any requested organism. For proteins that are to be expressed in more than one host, Eurogentec can create hybrid codon usage tables. Codons below the selected threshold in either host are replaced, and the frequencies for the remaining codons can be calculated using either the frequencies for the most restrictive organism or using the mean value for each codon.
| E | Y | D | H | | E | Y | D | H | | E | Y | D | H | | E | Y | D | H | | |
| TTT | 0.58 | 0.59 | 0.37 | 0.45 | TCT | 0.17 | 0.26 | 0.08 | 0.18 | TAT | 0.59 | 0.56 | 0.37 | 0.43 | TGT | 0.46 | 0.63 | 0.29 | 0.35 | | E: E.coli |
| TTC | 0.42 | 0.41 | 0.63 | 0.55 | TCC | 0.15 | 0.16 | 0.24 | 0.22 | TAC | 0.41 | 0.44 | 0.63 | 0.57 | TGC | 0.54 | 0.37 | 0.71 | 0.55 | | Y: Yeast |
| TTA | 0.14 | 0.28 | 0.05 | 0.07 | TCA | 0.14 | 0.21 | 0.09 | 0.15 | TAA | 0.61 | 0.48 | 0.42 | 0.28 | TGA | 0.30 | 0.29 | 0.26 | 0.52 | | D: Drosophila |
| TTG | 0.13 | 0.29 | 0.18 | 0.13 | TCG | 0.14 | 0.10 | 0.20 | 0.06 | TAG | 0.09 | 0.24 | 0.32 | 0.20 | TGG | 1.00 | 1.00 | 1.00 | 1.00 | | H:Human |
2. Eliminating unfavorable and uneven GC content: The strength of the guanine-cytosine bond can lead to undesirable mRNA secondary structures. The most effective candidate sequences will have more favorable GC content.
3. Avoiding unfavorable mRNA secondary structures: Overly stable mRNA secondary structures, particularly at the 5' end of the transcript, have been implicated in reduced gene expression. The potential of a transcribed mRNA to adopt such a structure can be identified using free energy calculations. Candidate sequences containing significant mRNA secondary structures can then be screened out.
4. Adding or removing restriction sites: The presence or absence of selected restriction sites is often important to facilitate subsequent gene manipulations such as swapping between vectors, exchanging protein domains, and adding or removing peptide tags or fusion partners. Candidate sequences can be tested to ensure the correct placement or elimination of restriction sites.
5. Other constraints: Additional constraints that can be used to filter the gene synthesis solutions include adding or removing polyadenylation signals and other regulatory elements, adding or removing immuno-stimulatory or immuno-suppressive elements (for DNA vaccines), RNA methylation signals, selenocystein incorporation signals and other factors, depending on the biological system used and specific application or concern.
6. Additional features of our gene design service: Manipulation of restriction enzyme cutting sites: These sites can be added or removed during the design process to facilitate future manipulation of the construct. Design of fusion proteins: Any epitope tag sequences (e.g, HIS tag and GST tag) or protease cutting site can be incorporated into the designed gene for the expession of a fusion protein. The delivered synthetic gene construct is ready for expression and purification without further manipulation. Creation of gene variants (mutant forms): Multiple variant forms (or variant library) can be designed for functional study and screening.
Applications
Eurogentec's custom gene synthesis technology is extremely versatile, applicable in many different kinds of procedures, such as cDNA production, heterogenous protein expression, and the creation of gene variants and recombinant antibodies. Custom gene synthesis comes with our optional codon optimization service, which can dramatically increase heterogeneous protein expression, at no additional charge.
Codon Optimization: Not all host expression systems share the same codon bias.The expression of, say, a human gene in E. coli can increase dramatically when the codon frequency of that gene is matched to that of the host system, but that is only the first step of the total gene optimization process.
Recombinant Antibodies: Human-mouse chimeric antibodies retain the specificity of the original murine variable region but assume the effector function of the human constant region, combining the high specificity of hybridoma antibodies with the mutability and reliable replication of plasmids.
cDNA Constructs: custom gene synthesis technology allows for the direct production of high-quality cDNAs for cDNA libraries and other purposes. Gene Synthesis completely eliminated the uncertainty of PCR cloning, and the cost is much less than tranditional PCR cloning.
Gene Variants and SNPs: Most alternative splice methods are unwieldy and can have unpredictable results. Custom gene synthesis incorporates rigorous regulation and verification of alternative splice products.
DNA Vaccines and Vectors: Since the production of an effective DNA vaccine involves not only the sequence itself but also the target, delivery system, expression, regulation, and inhibition of the gene therapy product.
Microarray-Ready cDNA: Reverse transcription bias and other problems can alter the relative amounts of cDNA measured by microarray assays. Eurogentec's advanced gene synthesis technology includes several means of countering these issues.