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It has become a hotspot and keystone in gene engineering and bioengineering to produce recombinant proteins through heterologous expression systems. Unfortunately, not all the genes could be successfully and effectively expressed in heterologous hosts. The role of gene itself in regulating translation process through its intrinsic sequence characteristics such as codon bias, codon pair bias, GC content, mRNA secondary structure and mRNA stability, has been gradually elucidated. Here we review these factors that influence the translation processes and their corresponding optimization methods in the process of gene design. We emphatically discussed codon bias and codon pair bias and their optimization methods. In particular, the latest theories of codon harmonization and codon pair harmonization were discussed and compared with the traditional codon and codon pair optimization strategies in gene design.

Gene synthesis is the most fundamental and widely used technique in biological research. The synthesis of DNA encoding regulatory elements, genes, pathways and entire genomes provides powerful ways to both test biological hypotheses and harness biology for our use. The emerging field of synthetic biology is generating insatiable demands for synthetic genes. And the past couple of years witnessed exciting new developments in microchip-based gene synthesis technologies. This review discusses the current methods of chemical DNA synthesis and gene assembly, as well as the latest engineering tools, technologies and trends which could potentially lead to breakthroughs in the development of accurate, low-cost and high-throughput gene synthesis technology. These new technologies are leading the field of synthetic biology to a higher level.

Constructing robust gene circuits is a fundamental work for synthetic biology. Bacteria with suicide gene circuit based on quorum-sensing will kill themselves in a controllable pattern upon certain cell density. In the media of different IPTG inducer concentration, we observed the growth and suicidal behavior of the Escherichia coli. Top10F' with such gene circuit, screened the mutants and determined their mutated loci. The results show that, with higher IPTG concentration, the more wild type bacteria were killed; as well the mutants emerged earlier and spread over the population more quickly. The sequence of plasmids in those mutants revealed that a transposon inserted into the luxR gene and therefore disrupted Quorum-Sensing of these individuals. Furthermore, the insertion sequence of the plasmid can solely result in the mutants escaping from suicide.

Objective: The Human papillomavirus type 18L1 (HPV18L1) gene was synthesized by overlapping PCR after optimization using plant preferred codons., Methods: The gene sequences of HPV18L1 were obtained from GenBank and analyzed using DNAMAN, Lasergene, Vector NTI and BLAST. The target sequence was selected and modified using plant preferred codons by the Synthetic Gene Designer and JCat (Java Codon Adaptation Tool) with the addition of a His-tag to the C-terminus to construct the full-length modified HPV18L1 (mHPV18L1). mHPV18L1 was divided into 5 large segments, namely LS1 to LS5, with sizes ranging from 204 to 477 bp. Forty-three small oligonucleotide fragments with sizes of 57-59 bp and 6 pairs of primers were designed and synthesized. mHPV18L1 was amplified by overlapping PCR and subcloned into pMD18-T vector. The recombinant plasmid was identified by restriction enzymes digestion and sequencing., Results: mHPV18L1 was successfully assembled using overlapping PCR. The results of digestion with restriction enzymes and PCR amplification confirmed that the recombinant vector pMD18T- mHPV18L1 contained the inserts with expected size of 1749 bp. mHPV18L1 sequence was confirmed by sequencing., Conclusion: mHPV18L1 with plant preferred codons and the recombinant vector pMD18T- mHPV18L1 have been obtained.

Objective: To develop a PCR-based method for gene assembly of tetanus toxin C fragment (TETC) DNA sequence from a large number of oligodeoxyribonucleotides (oligos)., Methods: To allow for its cloning and expression in Lactococcus lactis, the TETC gene sequence was designed according to the known TETC gene sequence (GenBank accession number M12739, 367-1719) and the amino acid coding in Lactococcus lactis. The sequence contained 1383 nucleotides (nt) with Sal I site added to its 5' end and Xho I and Hind III sites to its 3' end. There were 209 synonymous codon substitutions in the designed gene sequence as compared with the sequence reported in GenBank for amino acid coding in Lactococcus lactis and elimination of the restriction site of EcoR I and Kpn I. The 1380 nt of the sequence was divided into 68 oligos designated as TETC 1 to TETC 68, each containing 40 nt. A 16 nt oligos designated as TETC 69 was designed as the downstream primer. The TETC 1-24 fragment was acquired using the oligos TETC 1 to TETC 24 by PCR-based gene assembly method, and the TETC 23-46 and TETC45-68 fragments were assembled similarly. The full-length TETC gene was assembled using TETC 1 and TETC 69 as the primers when the 3 fragments were mixed. The target gene was gel-purified and digested with Sal I and Hind III, followed by ligation to the pBluescript II SK(+) and digestion with the same enzymes. The positive clones were confirmed by restriction enzyme excision and sequencing., Results: Three 500-bp fragments were acquired by PCR-based gene assembly, and the full-length TETC gene was obtained from the 3 fragment mixed at a equal concentration by a second PCR-based gene assembly using TETC 1 and TETC 69 as the primers. The target gene was cloned to pBluescript II SK(+) vector, and sequence analysis of the positive clones indicated that the assembled sequence was identical to the designed coding sequence of TETC gene., Conclusion: PCR-based assembly of the synthesized constitutive gene fragments into the complete sequence can be an effective strategy for synthesis of long DNA sequences in vitro.

Gene synthesis is gaining popularity as a versatile means of cloning genes. The prevailing synthesis methods are PCR-based and ligase-based gene synthesis. Both of them are not independent of the sequence content of the target gene. In the present study, a new gene synthesis method-isothermal unidirectional elongation method (IUEM) is proposed, which involves the cooperation of three enzymes, and is independent or partially independent of the target gene sequence by the specially designed oligonucleotides that form hairpins. The synthesis process is isothermal and serial in IUEM. Several factors that affect the elongation were studied, and two DNA segments of length 254 bp and 300 bp were synthesized with IUEM. The synthesized segments could work as building blocks for the synthesis of longer DNA sequences.

Transcriptions are regulated by transcription factors. Natural transcription factors usually consist of at least two functional domains: a DNA-binding domain and an effector domain. According to this, novel artificial transcription factors are designed to up or down regulate transcription and expression of a target gene. The Cys2-His2 zinc finger domain is a DNA-binding module that has been widely used as the DNA-binding domain in artificial transcription factors. Each zinc finger domain, which comprises about 30 amino acids that adopt a compact structure by chelating a zinc ion, typically functions by binding 3 base pairs of DNA sequence. Several zinc fingers linked together would bind proportionally longer DNA sequences. According to the "bipartite complementary" library strategy, a pair of zinc finger phage display libraries were constructed. After construction of the libraries, a 9bp sequence (5'-GCAGAGGCC-3') on the promoter of SV40 was chosen as a target for next step. After parallel selection, PCR amplification, desired fragments recovery, re-ligation, and additional rounds of selection, phage enzyme-linked ELISA experiments were performed to identify specific binding clones displaying the zinc fingers with predetermined sequence-specificity to our target sequence. Then two clones with strong ELISA signals were chosen to be tested for binding both to its full target site (5'-GCAGAGGCC-3') and to sites containing single transition mutations. The binding specificity of one of the two clones (clone 3) was shown to be fairly good. The three-finger DNA-binding domain targeted to SV40 promoter, that is, zinc finger sequences on clone 3, was fused to KOX1 suppression domain KRAB and cloned into pcDNA3.1 (+) (which expression product was artificial transcription factor). The zinc fingers (which expression product was the DNA-binding domain of artificial transcription factor) and KRAB domain only (which expression product was effector domain of artificial transcription factor) were also cloned separately into the same expression vector. All constructs contained an N-terminal nuclear localization signal. Every of the vectors (including pcDNA3.1 (+) without inserting sequences) were cotransfected with pGL3-Control and pRL-TK and the activity of luciferase was used to indicate the function of product from transfected expression vectors. Our artificial transcription factor was proved to repress the expression of reporter gene efficiently,while with only DNA-binding domain or effector domain the repression was not remarkable. By adding different effector domains and changing the DNA-binding domain, artificial transcription factor would have a wide range of potential applications.

The gene of LIP1, the most important isoenzyme of Candida rugosa lipase (CRL), was artificially synthesized according to its mature peptide sequence. It consisted of 20 codons of preference in Pichia pastoris. The artificial gene was cloned into methanol-inducible expression vector pPICZalphaA, and constitutive expression vector pGAPZalphaA, respectively. The linearized recombinant plasmids were transformed into chromosome of Pichia pastoris SMD1168H strain by electroporation. The abilities of expressing LIP1 in both transformed yeasts had been compared, and the yeast transformed with pGAPZalphaA was more efficient than pPICZalphaA. A recombinant yeast strain named CHT-II expressed LIP1 constitutively, and was the most efficient one. Some enzymatic properties of the recombinant LIP1 were also determined. CHT-II secreted LIP1 into supernate at a level of 2.00x10(5) u/L after 72 h (the cells had been transferred to fresh culture medium at the 24th h). After optimizing the conditions for high cell-density fermentation, the selected yeast strain could secrete LIP1 into supernate at a level of 1.395x10(6) u/L after 72 h. The results indicated that this modification of lip1 gene was successful.

Two new analog genes of the alpha-human atrial natriuretic polypeptide (alpha-hANP) were constructed by accurate intragenic recombination of analog RH-1 gene with ANP gene in vitro, both of which had been synthesized and reported. One recombined analog gene, RH-2, was designed to contain an equivalent of the alpha-hANP gene, capping the peptidase inhibitor SQ20881 sequence at its 5'end. The other analog gene, RH-3, was designed to contain an equivalent of the alpha-hANP gene, wedging in two proline codons CCG & CCG at the 3' end. The recombinants bearing the genes, bacteriophage RH-2/M13mp18 & RH-3/M13mp18, were identified by DNA domain combination analysis--in situ hybridization and dot blotting with three probes matching three specific DNA domains, and by restriction analysis. The DNA sequences of RH-2 and RH-3 were confirmed by the dideoxynucleotide chain termination method.