CHE.496/2009/Responses/a5

Engineering Principles

• Discussion leader: Thaddeus

Thaddeus's Response

Another side of genomics: Synthetic biology as a means for the exploitation of whole-genome sequence information

• DNA fabrication is a major component of synthetic biology. This paper describes the course of fabrication technology and where it will likely go in the future.
• In the past molecular biology has been largely done using recombinant DNA products as a source for new sequences of genes because it was cheaper and quicker.
• Synthetic genes are more flexible and do not rely on access to the physical gene of interest to be useful. Only sequence information is required to make a synthetic gene.
• Synthetic genes are made by synthesizing short oligonucleotides which are put together using ligase and PCR.
• Poliovirus genome was recently synthesized chemically in the absence of a template strand of DNA.
• Host with minimal set of genes is being generated as a line with minimal complexity of the interactions between natural and endogenous genes.
• Oligonucleotides are currently the most expensive part of DNA synthesis. DNA chip synthesizers are being developed which could severely reduce this associated cost.
• This article is useful for the VGEM team because as designers of DNA sequences it will be helpful to understand the fabrication process so that we can be mindful of the limitations and opportunities made available by current technology.

Codon bias and heterologous protein expression

• This paper deals with the issue of codon bias and the problem it causes when genes from one organism are expressed in a different organism with a different codon bias.
• The genetic code is redundant with each amino acid being coded for by and average of three codons.
• Any protein can be coded for by a huge number of sequences.
• Different organisms favor different codons for each amino acid so that tRNAs may be used more efficiently. Highly expressed genes are encoded by the most favored codons. Less expressed genes would include rarer codons.
• Some organisms differ from the conventional genetic code so their genes are inaccessible using standard recombination technology.
• One way to bypass rarity of tRNAs is up regulation of tRNA. Poor solution because alters cell metabolism and tRNA modification is disrupted.
• Better solution is resynthesis of gene using optimized codons.
• Allows matching with host genome as well as control for unfavorable gene structure.
• This article will be useful to us because we will be transferring a gene from one organism to a different organism. We will have to account for how codon bias will affect the translation of our gene product. It may be necessary for us to adjust our sequence to optimize expression based on codon frequency.

Thaddeus Webb 16:38, 22 February 2009 (EST)

Rohini's Response

Codon bias and heterologous protein expression

• Expression of proteins in heterologous hosts is a challenge because the proteins might have codons that are rarely used in the target host
• 1977-Genetech scientists produced the first human protein (somatostatin) in a bacterium

-They synthesized the 14 codon long somatostatin gene using oligonucleotides

• Presently, genes are cloned from cDNA libraries or from PCR
• Key concept: “the DNA sequence used to encode a protein in one organism is often quite different from the sequence that would be used to encode the same protein in another organism”
• The genetic code uses 61 codons to encode 20 amino acids and 3 codons to terminate translation
• “The degeneracy of the genetic code enables many alternative nucleic acid sequences to encode the same protein.”
• The E.coli bacterium is not the optimal host for expressing proteins encoded with human codons
• Codon usage is important in prokaryotic gene expression
• Improving protein expression by modifying the host can be done by expanding the intracellular tRNA pool of the host, however the over production of tRNA requires other cellular components that might be limited in supply

-A better strategy would be to alter the rare codons in the target gene so that they reflect the codon usage of the host, without disrupting the amino acid sequence of the encoded protein

• Interesting point: Viral codon optimization is performed for DNA vaccine research in order to increase the immunogenicity of the target
• Scientists are currently trying to find a way to efficiently allow for post-translational modification of the protein
• Main point: the design and use of synthetic genes can allow scientists to have better control of heterologous protein expression

Another side of genomics: Synthetic biology as a means for the exploitation of whole-genome sequence information

• Synthetic biology- use of biosystems founded on the chemical synthesis of the coding DNA
• Moore’s law- the complexity of an integrated circuit with respect to minimum component cost will double in about 24 months based on the rate of technological development
• Synthetic genes are useful in:

1) Re-engineering the target sequence 2) Making an efficient construction of a family of related, but different constructs

• The DNA in synthetic genes is comprised of short oligonucleotides
• New sources for oligonucleotides have been found thereby making the production of DNA synthesizers cheaper and efficient
• A method of amplifying the oligonucleotides has been used
• There are two methods of producing oligonucleotides:

a) Use of a microfluidic synthesizer platform b) Array-derived process

• Array derived oligonucleotides will become ‘standard reagents’ in the daily work of scientists

Rohini Manaktala 16:58, 22 February 2009 (EST)

Patrick's Response

• Another side of genomics: Synthetic biology as a means for the exploitation of whole-genome sequence information
  • This article is about the use and creation of synthetic genes. Synthetic genes provide an alternate way of obtaining natural DNA, which enables the ability to make novel genes. In order to have the ease of constructing biological systems, the cost for constructing the gene sequences that make that possible must be very low so that designing elaborate systems is within our means of production. In the days before DNA synthesis, researchers used DNA recombination, which was limited to the genes involved that were recombined. With DNA synthesis, we could theoretically create sequences that have never taken form on earth (how realistic that would be is another question). Gene sequences can be obtained from online sequence databases and used in synthetic gene design, which VGEM and other teams have done – for example the blue and orange fluorescent proteins came from genebank.
• Codon bias and heterologous protein expression
  • This article describes the function of codons in an organism, in which organisms use particular codons to encode polypeptide sequences, which affect the expression of gene vectors. Different organisms prefer different codons because different organisms have slightly different ribosomes, in which ribosomes are responsible for “translating” DNA to proteins in all biological organisms through the catalysis of individual amino acids into the polypeptide sequence. The main issue with inserting foreign DNA into a chassis based on another biological organism is that of rare codons. Sometimes there are gene sequences in plants or otherwise that have codons that are not present in e. coli or whichever chassis we use. As a result, even if we are successful in building and transforming our vector into e. coli, the plasmid may not work due to a rare codon that makes completing the polypeptide sequence impossible to form. As a result, gene sequences are always optimized to remove rare codons that could damage the chances for expression in the chassis. The VGEM 2008 team used _Gene Designer_ software from DNA 2.0 to do the optimizing of our bioplastic sequences.
• Patrick Gildea 05:16, 23 February 2009 (EST):

Joe's Response

• Another side of genomics: Synthetic biology as a means for the exploitation of whole-genome sequence information
  • The creation of synthetic genes is based on the fact that target DNA is produced by programmed chemical synthesis of short oligonucleotides
  • for synthetic synthesizing of DNA: The complete sequence can be synthesized, and the backbone ligated or using a DNA polymerase enzyme to fill in part of the gaps in the sequence.
  • A huge hindrance is the source of oligonucleotides, but there are several promising sources.
  • microarray-based DNA synthesis is cost effective and may revolutionize the field of synthetic biology.
  • Using array-derived oligonucleotides with PCR. This provides a source of coding oligonucleotides which will also reduce development costs.
  • Optimization and fine tuning is still required
  • Longer sequences will be synthesized and utilized in innovative applications.
  • array-derived oligonucleotides will be optimized and become a standard tool of researchers.
  • Hopefully, we get the benefit of even lower costs and optimization.

• Codon bias and heterologous protein expression
  • Difficult to express proteins in heterologous hosts since the different ribosomes will present the codon bias problem
  • (A codon consists of three DNA bases)
  • A codon adaptation index has been defined based on the correlation between codon bias of a gene and the expression of its protein
  • Major problem because the genetic sequence that works for expressing a protein in one organism may be different for another organism for the same protein. (Rarer tRNAs per organism.)
  • Expression can be improved by modifying the host by overexpressing the genes that encode the rare tRNAs. However, this decreases any tRNA modification and creates nonsense errors in translation.
  • Another solution is to alter the rare codons in the target gene so that they more closely reflect the codon usage of the host. (By site-directed mutagenesis or resynthesizing the entire gene.)
  • We will have to optimize our DNA sequences to account for codon bias when we transfer our DNA to the heterologous host.

Joe Bozzay 13:01, 23 February 2009 (EST)

Maria

Another side of genomics: Synthetic biology as a means for the exploitation of whole-genome sequence information

• Synthetic gene: oligonucleotides (25-70 bases) are extended with PCR
• synthesize complete sequence: ligate backbone
• or synthesize in segments: finish with DNA polymerase
  • cheaper: use less synthetic oligonucleotides
  • but higher risk for mutation
• synthesis of poliovirus cDNA
• recreation of a working virus from “sequence stored in silico”=>possible bioterror
• existing problem: quick/cheap source of oligonucleotides
  • use of arrays is possible technological development

Codon

• most synthetic genes are copid from cDNA libraries
• de novo construction: high cost, time, effort
• central dogma does not cross species
• index: measures of degree of preference for a codon
• Ecoli not ideal for human proteins: differing codon profiles
• different tRNA levels

• Maria Fini 18:27, 23 February 2009 (EST):