CHE.496/2008/Responses/a5

Natural biological parts

• Discussion leader: Patrick Gildea (Discussion guide)

Kevin Hershey's Response

• Another side of genomics: Synthetic biology as a means for the exploitation of whole-genome sequence information
  • Stahler's main focus in this paper is on the development of in situ microarray analysis. Such examples of these array-derived oligos include the Maskless Array Synthesizer and the use of a microfluidic synthesiser platform. He discusses how this new advancement will allow for cheaper and quicker synthetic biology. However, Stahler's definition of synthetic biology is different from the previous papers discussed in this class. He defines synthetic biology simply as "... the making and use of biosystems founded on the synthesis of the coding DNA (and potentially RNA) based upon chemically synthesised nucleic acids oligonucleotides of programmable sequences." However, even though his definition is different than ours, it is still a useful tool to be used in the practice of synthetic biology for the iGEM competition.
• Codon bias and heterologous protein expression
  • Gustaffson's paper deals with codon bias in organisms. In brief, different organisms have a bias towards codons for protein expression. For example, while two different codons may code for one amino acid, they may each work better in different organisms. While Gustaffson points out some areas in which care should be taken (i.e. repetitive sequences), companies such as DNA2.0 offer free software to easily complete codon optimization.
• KPHershey 22:26, 29 January 2008 (CST)

Eyad Lababidi's Responses

• Another side of genomics
  • This raeding was pretty dense with biological terms and processes that i had to ask my bio friends for explanation, but the gist i got from the reading is that the field has had recent breakthroughs that will lead to cheaper and more powerful synthesis opportunity. Oligonucleotides being one of the cost constraints apparently there have been break throughs to more easily produce desired oligonucleotides.

• Codon bias
  • Its really interesting to know that we now have the ability to line up codons in any order we want because it truly gives us the ability to create our own brand new unique cells whether they work or not. Although its surprising we have this power its surprising that we dont really understand why certain codon chains produce more reliable and more expresses proteins, it almost seems like we are playing with science that could have bigger repercussions then we understand. although it does seem quite simple when its broken down to a cookie cutter recipe of how to more successfully produce proteins in your cell.
• Eyad Lababidi 12:50, 30 January 2008 (CST)

George Washington's Responses

• Another side of genomics: Synthetic biology as a means for the exploitation of whole-genome sequence information
  • For the past 40 years, DNA recombination technology has been one of the primary tools used in developing biotechnology and investigating biological systems. The difficulty with this is that recombination is limited, in that it can only make use of genes that nature provides us, with little room for new development outside of discovering new organisms. Thus, there has always been some need for gene synthesis, the artificial construction of genes when they could not otherwise be obtained, either due to logistics or prior non-existence. In recent years, though, this synthesis technology has grown manifold, with progress in the development and ligation of short oligonucleotides, especially in microarrays that permit immense parallelism. This has great relevance for our team, as this is directly related to the exponential drops in price that we've been witnessing in the DNA synthesis market; the cheaper it is to obtain our DNA, the more grand in scale our projects can be. However, one of the dangers of such large scale DNA synthesis is that QA is still not incredible, with quoted rates of one error per 1,394 bp, although this has probably improved in the intervening couple years.

• Codon bias and heterologous protein expression
  • This article describes how which codons an organism uses to encode polypeptide sequence affect expression of foreign gene insertions. If we need to insert a new gene in an organism, we should first take into account the codon frequencies present in the original organism. We should try to adjust the codon frequency in the desired gene such that it matches that in the original organism, while avoiding rare codons. This ensures that tRNA necessary to translate the protein is available in necessary quantity in the cell. Of course, there are numerous other considerations to make when designing a gene for inclusion. Among these are the avoidance/creation of restriction sites, the reduction of repetition in the sequence, which can lead to undesirable secondary structure, reducing translation efficacy, and the prevention of GC overabundance. In sum, there are many factors to take into account before inserting a gene into a new host; it must be specifically designed to thrive in the new host by matching various host factors.

• George Washington 15:08, 30 January 2008 (CST)

Brandon Freshcorn's Responses

• Codon bias and heterologous protein expression
  • This article points out the problem that there is still a significant underlying problem in biotechnology- the way a DNA sequence is expressed in one organism is quite different from the sequence another organism would use to describe the same protein. This leads to a low expression, or even no expression at all, of the described protein. Gustafsson et al. describe remedies for this problem of codon bias, such as increasing the intracellular tRNA pool (by over-expressing genes that encode the rare tRNAs). However this carries, among other problems, the problem of the actual logistics of manipulating the genes responsible for tRNA. An alternative is to optimize the sequencing of the DNA so that codons that are rarely seen by the organism are avoided, and a codon more common to that particular organism is used.

• Another side of genomics: Synthetic biology as a means for the exploitation of whole-genome sequence information
  • Stahler et al. discuss in this review the rise of array-derived oligonucleotides and its power as a tool for synthetic biology coupled with its decrease in price. He also predicts the future of synthetic biology will lie in two trends: synthesis of longer sequences, and the optimization of quality of array-derived nucleotides. This latter trend does remind me of one concern though. The better quality oligonucleotide sequences contain one error per 1394 bp. I assume the author is referring to a point mutation (as a deleted base pair would lead to a frame shift and be disastrous). However, this could still prove to be a problem for our own project, as that one point mutation might code for the wrong amino acid and alter the structure of our protein. This is something we shall have to remain alert to.

• --Brandon S. Freshcorn 16:37, 30 January 2008 (CST)

Dan Tarjan's Responses

• Synthetic biology as a means for ....
  • This article highlights the advances being made in reducing the cost of snythesizing physical DNA. The current efforts they discuss are focusing on oligonucleotides and automated methods that can ligate them into longer DNA strands. While the reduction in cost for current DNA synthesis companies will certain result from the adoption of quicker, more efficient and less error-prone technologies I think the real advance will happen when labs have a table-top DNA synthesis machine. This development will allow the gap that the paper refers to between the availability of sequence information and the utilization thereof, to close.

• Codon bias...
  • Optimizing genetic code for heterologous expression is a necessary tool in the synthetic biologist's repertoire as the idea of synbio revolves being able to move pieces of genetic code between organisms to do useful things. This article introduces the concept and shows why it is necessary. It goes into the reasons WHY expressing a non-native gene in an organism runs into problems - tRNA concentrations and such. However the authors also highlight the shortcomings with initial attempts to try to surpass these roadblocks. Despire the difficulties involved, the authors have found that various optimization techniques yield drastic results and list them in the table. Challenges that still lie ahead for the optimization effort include short-comings in a host's post-translation environment that are necessary for proper protein folding.

• Daniel R Tarjan 16:28, 30 January 2008 (CST)

George McArthur's Response

• Another side of genomics: Synthetic biology as a means for the exploitation of whole-genome sequence information
  • This article predicts that within 10-20 years, synthetic genes will be the tools of choice in the molecular biology laboratory. Synthetic genes provide researchers a way of obtaining natural DNA that is difficult to come by and a way to make completely novel genes. These man-made genes constitutes a true synthetic biology. However, valuable parts can be obtained from online sequence databases and used in synthetic gene design, which many iGEMs teams in the past have done. The majority of the article discusses methods of cheaply and abundantly producing synthetic DNA (e.g., in situ microarray synthesis). We are now able to truly write genetic code. The big question is what should we write.
• Codon bias and heterologous protein expression
  • Different organisms prefer different codons because different organisms have slightly unique ribosomes. As we know, many alternative nucleic acid sequences encode the same protein. Any given protein can be expressed at high or low levels depending on how efficient the ribosome is at reading the sequence and cranking out amino acids. If we know which codons an organism prefers (i.e., will express high levels of that protein), then we should optimize the sequence accordingly so that we have an efficient process. There are tools that help us do this quickly. DNA 2.0 has developed Gene Designer, which optimizes nucleotide sequences for a set of well understood organisms (including E. coli).
• GMcArthurIV 16:51, 30 January 2008 (CST)