Natural biological parts
- Intro to Gene Designer (George)
- Discussion leader: Patrick
- Another side of genomics: Synthetic biology as a means for the exploitation of whole-genome sequence information link
- Purpose: The paper reviews the area of Synthetic Biology – the design/development of biosystems founded on the synthesis of DNA as well as recent developments in the field.
- Background material is given in the various fields and technologies (synthetic genes, DNA recombination tech, PCR and so on).
- The process for creating a synthetic gene: target DNA is produced by programmed chemical synthesis of short oligonucleotides where longer DNA molecules are formed together via ligation, PCR, and doing either of the following:
- Synthesizing the complete sequence and ligating the backbone
- Synthesizing part of the sequence, leaving gaps in the fragile hybrid and filling in the gaps with DNA polymerase enzymes
- In situ microarray synthesis represents a cost-effective technology that can combine pieces of DNA to form larger pieces of DNA in a cheaper and faster manner than older technologies.
- Conclusion: development costs of synthetic genes will drop and new applications will emerge such as novel self-replicating systems. Furthermore, more attention can be focused on downstream processing – optimization and fine-tuning (i.e. reduce the errors in the DNA “code” using mutation detection proteins). Further developments will result in longer sequences being synthesized and array-derived oligonucleotides will continue to be optimized in quality and performance.
- Codon bias and heterologous protein expression link
- Purpose: Discusses the fact that proteins are difficult to express outside their own original genome because they contain codons that interfere with the implanted host, non-canonical code in different organisms, or contain expression limiting regulatory elements.
- In the field of creating synthetic genes, the most trouble comes from the stage where the amplified gene is cloned into an expression vector, where the protein is not expressed or at very low levels. REASON: DNA sequences vary in differing organisms.
- Codon Bias - affects protein expression through optimizing the translational system and balance codon concentration with tRNA concentration
- Improve protein expression by modifying the host - overexpressing the genes encoding the rare tRNA's
- Codon Optimization: the more codons that a gene contains that are rarely used in the expression host, the less likely that the heterologous protein will be expressed at reasonable levels. So alter the rare codons in the target gene so that they may more closely reflect the codon usage of the host via sequential site-directed mutagenesis or resynthesis of the whole gene.
- Codon Bias is not the only factor that affects a gene’s expression, expression vectors and transcriptional promoters matter. As well as the efficient translation of the strands of DNA, where the polypeptide chain must fold right.
- Box 1 describes a gene design process to encode a specific protein through using an initial codon usage table and narrowing the possibilities from there (page 351)
We have learned about how important DNA synthesis is toward the development of synthetic biology. In addition, we have learned a basic methodology of the gene design process. I think the best way to go about ending the meeting is to discuss the gene design process that the VGEM 2007 team went through. Their rationale in selecting the codon (eliminating codon bias and unfavorable pairs), eliminate repeats and mRNA structures, add/remove restriction sites, *other constraints*, and then move into the area of the wet lab.