MICR3003 Molecular Microbiology  Lecture 2

Construction of Designer Bacteria

Dr J M Pemberton 2003

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Isolation of Microbes Which over-produce Antibiotics

Overproducer Colony With a zone of Inhibition around it

Antibiotic Sensitive Tester Strain S.aureus

HISTORY
1. Use of intensive and repeated mutagenesis of the fungus Penicilium during the 1940s lead to the isolation of strains which produce a hundred times more Penicillin G than the wild type strains. Penicillin G was active only against Gram Positive bacteria Semi-synthetic penicillin antibiotics such as Ampicillin and Carbenicillin have been developed which are active against Gram Negative bacteria. 2. Proteases, amylases, cellulases and a wide range of enzymes and metabolites have been produced from bacterial strains which were selected for overproduction after mutagenesis. Since GENE CLONING was invented in the early 1970s a rational approach has been used to construct strains of bacteria of use in Medicine, Agriculture and Industry. These strain constructions have been made much easier by the fact that many multigene phenotypes such as antibiotic synthesis are encoded in a single gene cluster.

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Is it Possible to Create a Synthetic Designer Bacterium?

Carl Zimmer. SCIENCE 14th Feb 2003 Vol299;1006-1007

Venter Cooks Up a Synthetic Genome in Record Time

Elizabeth Pennisi Science Volume 302, Number 5649, Issue of 21 Nov 2003, p. 1307.

The U.S. Department of Energy (DOE) announced last week that sequencing maverick J. Craig Venter had taken just 2 weeks to build a viral genome from scratch, Secretary of Energy Spencer Abraham predicted that it could lead to the creation of microbes tailored to deal with pollution or excess carbon dioxide or even to meet future fuel needs. "I didn't think it was a big deal," says Ian Molineux, a molecular biologist at the University of Texas, Austin. And Richard Ebright, a molecular biologist at Rutgers University in Piscataway, New Jersey, agrees: "This is strictly a limited incremental advance over current technologies." The skeptics focus on how hard it will be to go beyond the initial step, while Venter, head of the Institute for Biological Energy Alternatives (IBEA) in Rockville, Maryland, and former president of Celera Genomics, and his backers are proud to have gotten this far. All are in agreement, however, that the experiment demonstrated speed in converting raw ingredients into a functioning virus. The genome synthesized by the Venter-led group belongs to a bacterial virus, called a phage; when it was tested in a lifelike situation, Venter reported, it infected and killed bacteria just as natural phages would. Because his team stitched together the phage's DNA in just a few weeks instead of years, molecular virologist Eckard Wimmer of the State University of New York, Stony Brook, called the effort "a very smart piece of work."
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Venter's lab isn't the rst to stitch together an articial genome.

Molecular biologists have been trying to do this ever since they started generating the entire sequences of organisms. Last year, Wimmer and his colleagues assembled the 7000-base poliovirus genome from small pieces of synthesized DNA. And they made headlines when they showed that the virus was active (Science, 9 August 2002, p. 1016). But the task took 3 years to nish. This summer, Venter set out to do better. His team included IBEA collaborators Hamilton Smith and Cynthia Pfannkoch, and Clyde Hutchison of the University of North Carolina, Chapel Hill. Like Wimmer, they started with short pieces of DNA, pieced them together by matching up overlapping ends, and eventually generated a complete 5400-base-pair phage genome. Their approach differed from Wimmer's, however. They modied and added steps to speed the sequence's assembly and to make it more accurate. The work is in press in the Proceedings of the National Academy of Sciences. And Venter is convinced that he can build genomes 300,000 bases or longer. But even with these improvements, skeptics and supporters aren't sure how well the procedure will work for organisms with larger genomes. "Going from a phage to a microbial genome to having a microbe that's synthetic is a very major step," says Patrinos. But he thinks it's worth betting on.

THE CANDIDATES
Mycoplasma genitalium smallest,free living microbe\ 0.2-0.3 mm in diameter No cell wall Low G+C(25-40%) 580 kb Genome 517Genes Escherichia coliK12 Most intensively studied and used microbe Physiology and genetics well known to most molecular biologists Rod shaped 1.5 X 3.0 mm Cell wall 50% G+C 4750 kb Genome 4000 genes

CHARACTERISTICS OF A DESIGNER BACTERIUM

The Hardware-The Core Bacterium a small genome like the Mycoplasmas the growth rate and utility of E.coli K12 so familiar to most molecular biologists The Software- Gene Clusters Encoding Desrired Phenotypes Production of Hydrogen as a cheap and unlimited energy source Synthesis of antibiotics and other chemotherapeutic agents Degradation and recycling of pollutants
Etc

CONSTRUCTION OF THE CORE BACTERIAL HARDWARE A. Creating Life?- Synthesise the Genome From Scratch An estimate made by Venter and co-workers is that the Mycoplasma genome(580 kb) can be cut down from 480 genes to 250-350 genes and still be a functional free living organism. How to resolve this question? 1 Synthesise the entire genome(580 kb) from scratch without sequence errors. A major task since synthesis of the Polio Virus (7.5 kb) had a number of sequence errors Remove the nucleus from a normal Mycoplasma cell and insert the test-tube genome and see if bacterium comes to life-An unknown. or Transform synthetic genome into E.coli and at cell division two different bacteria should arise from a single transformant.
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3 A worthwhile challenge in itself. Venter estimates the project will take 3 years.

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Challenges/Problems
Polio Virus was the rst genome synthesised- 7,500 bp Not a living organism Of the 517 Mycoplasma genes, 30% have unknown function. The best answer is sequential deletion of each gene in a targeted Way. The technique to do this is not available. An opportunity for a budding scientist?

Mycoplasmas are difcult to grow, miserable to work with and their growth rate would need to be speeded up for them to be the basis of the designer bacterium hardware

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B. Use of a pre-existing free living bacterium

E.coli K12 or a Redesigned E.coli with a smaller genome 1 E.coli K12 is the workhorse of molecular biology 2 E.coli K12 is easy to grow and manipulate
3 4. The genetics and physiology of E.coli K12 is familiar to most molecular biologists It may be necessary to develop a technique to delete non-essential genomic DNA from E.coli K12.But will the loss of genes mean a loss in amenability e.g. the strain may not grow well. Could a synthetic E.coli be constructed using the homologs of the 480 Mycoplasma genes.

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6. E.coli is known to express such complex multi-gene phenotypes such as: Antibiotic synthesis-violacein (1st) rebeccamycin(2nd) staurosporine (3rd?) polyketides ? Carotenoid biosynthesis Nitrogen xation

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C. Isolate A New,Very Small, Bacterial Genome

1. 1. 3. 4. It is easy to transform E.coli with large molecules of DNA An example would be BACs or F-primes Techniques are available to harvest large DNA moleculs from the biosphere. Could some of these molecules be the entire genomes of new bacteria? What is certain, but yet to be discovered, is that two different bacterial genomes can exist in the same cell. Yet they can exist as separate bacteria. A type of non-obligate symbiosis. Many bacteria have more than one main chromosome Could one of these entire main chromosomes be deleted and the cell remain viable
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D. Reduction of compartmentalised genomes

Pseudomonas putida and P.aeruginosa have compartmentalised genomes

They have a continuous segment of their main chromosome which contains all the essential functions. The rest of the main chromosome appears to encode non-essential functions

Non-Core Section

Core Section

Core Section

Non-Core Section

P.putida

P.aeruginosa
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HARWARE

SOFTWARE

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Antibiotic Gene Cluster

Bacterium With Minimal Core Genome

Designer Antibiotic Producer

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Electronmicrograph of DNA

Designer Chromosome and the Additional Genes

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DESIGNER BACTERIA PRODUCING A VARIETY OF ANTIBIOTICS

Production of a Wide Range of Antibiotics in a Designer E.coli.

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