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University of Alberta - BioBytes

Why build a minimal genome?

Genomes are complex! Determining how simplified a genome can become enriches our understanding the function and interactions of cellular components. Simplified cells can be used as a well characterized chasses for synthetic biology. Moreover, a simplified cell can be used to study cellular processes in a controlled, characterized genetic background. Finally, developing a minimal genome requires us to develop and optimize molecular methods of genome assembly. These methods can be then applied to other high through put biology.

Presentations

The size and complexity of the genome make bioinformatics analysis essential. We used bioinformatics to accomplish the following:

- review lists of essential genes in the literature and existing databases and compile a preliminary essential gene list

- model the metabolic reactions and net growth rate of E.coli with given gene sets. This identified additional metabolic genes essential to a minimal genome.

- identify knock out combinations that could be tested in the wet lab, to verify the accuracy of our metabolic model.

- select standardized promoters and terminators that would replace the natural promoters and terminators of essential genes.

- determine which promoter should be used with which gene, by analyzing expression level data.

- design primers to amplify all essential genes from genomic DNA.

These steps have all been completed, and are described on the following pages.

REcoli Human Practices Component: High School Outreach

Our Human Practices component consists of presentations and pamphlets for high school and junior high school students about synthetic biology, iGEM, and our project. We’re doing this outreach primarily for the high school students’ own interest and benefit. Synthetic biology is rapidly changing the biotechnology industry, and an understanding of synthetic biology would enrich a student’s consideration of career choices. Becoming excited about a potential career option can also provide motivation for academic success. Our outreach is also a service to the research community, as the more students who know about synthetic biology, the more who may pursue it as a career. A greater pool of human resources can increase the quantity and quality of research

We believe that sharing one’s research with the community is an important responsibility, both to keep research accountable to the public and to open doors for the next generation to get involved. Even for those students who don’t pursue science, an exposure to what synthetic biology is allows them to make more informed, responsible choices as consumers and voters. Thus, through high school outreach, we’re setting a good example of good example of corporate social responsibility.

Finally, we strive to learn how to better communicate synthetic biology to students. In order to evaluate the impact of our outreach, we collect feedback forms after presentations and have an online survey.

Constructing of a Preliminary Essential Gene List from the Literature

Criteria for Gene Selection:

o Genes must be present in more than one literature list unless there is particular reason to suspect they are essential

o The REcoli metabolism is modeled after the minimal metabolism proposed by Gil et al 04, with the addition of cell wall, fatty acid, heme and ubiquitin synthesis, as Gil assumed these would not be necessary in a mycoplasma like minimal cell.

o Additional genes required for metabolism were selected based on pathway information in the Ecocyc database. Redundancy of pathways is likely why these genes don’t appear essential in Baba, Gerdes and PEC.

o Antitoxin genes are not essential as toxin genes would not be present

Genes for the following processes were included:

o DNA replication and cell division, but no DNA repair

o Chaperones, but no heat shock or membrane stress response system

o Transcription

o Translation

o Glycolysis

o lactate production from pyruvate to regenerate NADH

o PMF generation via an ATP synthase consuming ATP to export protons.

o Synthesis of acetyl-CoA from pyruvate

o Fatty acid synthesis

o Methylerithritol pathway (for undecaprenyl phosphate and a ubiquinone side chain)

o Synthesis of phosphatidylethanolamine, but no other phospholipids

o Pentose phosphate pathway (converts 6 or 3 carbon sugars to 5C sugars, such as ones needed in nucleotide biosynthesis)

o Lipoprotein synthesis (Int and lolB are lipoproteins and essential)

o Synthesis of nucleotides (deoxy and oxy) from nucleosides

o Attaching lipid and biotin groups to protein

o Transport:

PTC transport system (imports and phosphorylates glucose)

Inorganic phosphate transport

Nucleoside transport

Sec system (exports proteins to periplasm), including SRP for cotranslational membrane insertion. secB chaperone does not appear essential. There is NO tat system, which would be used to export cofactor containing folded proteins.

Lipoprotein transport to outermembrane

Glutathione transport

o Cofactor synthesis:

Riboflavin from GTP and ribulose-5-phosphate

FAD from riboflavin

NAD from nicotinamide

NADPH from NAD

CoA from pantothenic acid

Methylene tetrahydroxyfolate (mTHF) from folic acid

S-adenosylmethionine (SAM) from methionine

Thiamine diphosphate (TPP) from thiamine

Pyridoxal-5-phosphate (PP) from pyridoxal

Heme from glutamate

Ubiquinone

RNAs:

The rrnC operon supplies 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. At least one tRNA was included for each amino acid.

The complete list of essential RNA’s can be found here .

Statistics on BioBytes Gene List Based on Literature Review

Total 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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-<b>RNAs:</b>
+<h3>RNAs:</h3>
 <P>The rrnC operon supplies 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.  At least one tRNA was included for each amino acid.  </P>
-<P>The complete list of essential RNA’s can be found here. </P>
+<P>The complete list of essential RNA’s can be found <a href="http://SDfjfdkgjslkgd.ppt"> here </a>. </P>
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Team:Alberta/Project/Bioinformatics