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Team:Alberta/Project/Gene Selection
From 2009.igem.org
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Literature ReviewThere were four primary literature sources which were used for the determination of the essential genome. These genes were analyzed to construct a preliminary essential gene list Literature Gene List Data
Each gene list was determined in a variety of ways and there results show very little consistency. The number of genes from each source varies greatly. When the lists are compared to one another, there is very little overlap noted. Please see below: Venn Diagram of the Number of Essential Genes Shared Between Lists in the Literature The maximum number of genes in common between any two literature lists is 205, which is between Baba et al 2006 and Gerdes et al 2003. Only 48 genes were present in all four lists. The lack of consensus between these literature lists makes it very unreliable to use these genes in an essential genome. Still, these lists provide an important foundation for basic components that are required in an essential genome list. |
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Constructing the BioBytes Preliminary Essential Gene ListThe preliminary essential gene list is based on literature sources. As described in the modeling section of this wiki, the metabolic genes from this preliminary list were used as a starting point for the computer model and were greatly altered based on the model's suggestions. The following criteria were used for selecting genes from a literature source:
The basic functional groups of genes which were selected can be seen in the next chart and a detailed list of processes which were included follow. Essential Gene Functions Genes for the following processes were included:
RNAs:The rrnC operon supplies all the rRNA’s and three of the tRNAs. This operon was selected because it includes the great number of tRNA’s. To select the other tRNA’s, all tRNA’s listed as essential in PEC were first included. One tRNA was then selected for each anticodon that differed on one of the last two bases. Differences in the first base can be accommodated by anticodon 'wobble'. At least one tRNA was included for each amino acid. The complete list of essential RNA’s can be found here . |
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Statistics on BioBytes Preliminary Essential Gene ListTotal genes in Ecoli: 4762 Total protein coding genes in BioBytes preliminary essentials list: 332 Total number of RNA genes in BioBytes preliminary essentials list: 29 |
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Modeling GenesSelection of the individual modeling genes can be seen under the Modeling tab of the Bioinformatics section. From the lists determined by the model there are 116 genes where were determined to be essential. The model only contains metabolic genes of the MG1655 ''E. coli'' genome therefore all other types of genes were solely determined using literature sources. Many of the genes that were determined to be essential are due to the complex nature of metabolic pathways. It is not sufficient to simply delete a single gene and determine if the organism is viable. Often genes act in complexes, or become essential if other genes become deleted (for example in redundant processes where 2 genes fulfill the same essential function) allowing the modeling work to fill the gaps of numerous genes which are required for life. The function of many genes which were added include transport of small metabolic compounds. Although there are some new pathways that are added, the majority of the genes collected add to many of the pathways determined to be essential via the literature search. This shows that our list contained many of the correct pathways, just further research was required to determine all of the essential genes.
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BioBytes Final Essential Gene List
A final list of essential genes was produced from the literature review and the computer model.
Number of genes in list created from literature: 332 Number of additional genes suggested by model: 116 Number of genes in final essential genes list: 448 Number of genes in our essential gene list not classified as essential in the literature: 117 To view the complete list of Literature Genes click here . To view the complete list of Metabolic Model genes, click here .When these lists are compared to the literature lists of essential genes, they are found to have a very limited amount of overlap. In fact,our BioBytes Essential Gene List differs by 40%. Correlation of BioBytes Essential Gene List to Literature Lists  This list gives a much greater chance of success in producing a minimal genome than many of the sources that are presently available. With this list completed, our BioBytes approach can be used to assemble these genes into constructs and eventually produce the genome. Together, our modeling work along with BioBytes serve as a genome construction toolkit which anyone can use.
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Standardization of Gene Regulation ComponentsIn order to produce a well characterized and standardized minimal genome, numerous components have been standardized including promoters, terminators, and RBS sites. These have been incorporated into the BioBytes system either as individual parts (as in the case of promoters and terminators) or as components of our unique plasmids pAB and pBA (which occurred with the RBS site). Microarray data was also used to identify the relative amount of transcript which was produced by each essential gene and therefore which promoter to incorporate with each gene.
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Incorporation Into pAB/pBAIn order to produce the minimal genome, each individual gene is required to be amplified. In order to accomplish this, PCR was used to produce genes with distinct ends allowing for insertion into the pAB or pBA plasmids used in genome construction. 188 of these primers have been tested and added to the parts registry (please see the Achievements section for the parts list). |
