MICR3003 Lecture 2 Designer BacteriaDr J M Pemberton 2003

8/4/2019 MICR3003 Lecture 2 Designer BacteriaDr J M Pemberton 2003

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MICR3003

Molecular Microbiology

Lecture 2Construction of Designer Bacteria

-15Dr J M Pemberton 2003


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Isolation of MicrobesWhich over-produceAntibiotics

Overproducer ColonyWith a zone ofInhibition around it

Antibiotic SensitiveTester StrainS.aureus


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HISTORY1. 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 foroverproduction after mutagenesis.3. Since GENE CLONING was invented in the early 1970s a rational approachhas been used to construct strains of bacteria of use in Medicine, Agricultureand Industry.4. These strain constructions have been made much easier by the fact thatmany multigene phenotypes such as antibiotic synthesis are encodedin a single gene cluster.


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

Is it Possible to Create a Synthetic Designer Bacterium?


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Venter Cooks Up a Synthetic Genome in Record TimeElizabeth PennisiScienceVolume 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 first to stitch together an artificial 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 finish.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 differedfrom Wimmer's, however. They modified 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.


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THE CANDIDATESMycoplasma genitaliumsmallest,free living microbe\0.2-0.3 mm in diameterNo cell wallLow G+C(25-40%)580 kb Genome517GenesEscherichia coliK12Most intensively studied and used microbe

Physiology and genetics well known to most molecularbiologistsRod shaped 1.5 X 3.0 mmCell wall50% G+C4750 kb Genome4000 genes


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CHARACTERISTICS OF A DESIGNER BACTERIUMThe Hardware-The Core Bacterium a small genome like the Mycoplasmas the growth rate and utility of E.coli K12 so familiar to most molecular biologistsThe 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 pollutantsEtc


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CONSTRUCTION OF THE CORE BACTERIAL HARDWAREA. 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 errors2 Remove the nucleus from a normal Mycoplasma cell andinsert the test-tube genome and see if bacterium comesto life-An unknown. or Transform synthetic genome into E.coli and at cell divisiontwo different bacteria should arise from a single transformant.


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3 A worthwhile challenge in itself. Venter estimates the projectwill take 3 years.


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Challenges/ProblemsPolio Virus was the first genome synthesised- 7,500 bp Not a living organismOf the 517 Mycoplasmagenes, 30% have unknown function.The best answer is sequential deletion of each gene in a targetedWay. The technique to do this is not available. An opportunityfor a budding scientist?Mycoplasmasare difficult to grow, miserable to work with and their growth rate would need to be speeded up for them tobe the basis of the designer bacterium hardware


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B. Use of a pre-existing free living bacteriumE.coli K12 or a Redesigned E.coli with a smaller genome

1 E.coli K12 is the workhorse of molecular biology2 E.coli K12 is easy to grow and manipulate3 The genetics and physiology of E.coli K12 is familiar to mostmolecular biologists4. 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 notgrow well. 5. 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-genephenotypes such as:Antibiotic synthesis-violacein (1st)rebeccamycin(2nd)staurosporine (3rd?)polyketides ?Carotenoid biosynthesisNitrogen fixation


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C. Isolate A New,Very Small, Bacterial Genome1. It is easy to transform E.coli with large molecules of DNA

An example would be BACs or F-primes

1. Techniques are available to harvest large DNA moleculsfrom the biosphere.

3. Could some of these molecules be the entire genomesof new bacteria?

4. What is certain, but yet to be discovered, is that twodifferent bacterial genomes can exist in the same cell.

Yet they can exist as separate bacteria.A type of non-obligate symbiosis.

5. Many bacteria have more than one main chromosome6. Could one of these entire main chromosomes be deleted

and the cell remain viable


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D. Reduction of compartmentalised genomesPseudomonas putida and P.aeruginosa have compartmentalisedgenomesThey have a continuous segment of their main chromosomewhich contains all the essential functions. The rest of themain chromosome appears to encode non-essential functions

P.putida P.aeruginosa

Core SectionNon-Core Section Non-Core SectionCore Section


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+

Designer Antibiotic Producer

Bacterium With Minimal Core Genome

Antibiotic Gene Cluster

HARWARE SOFTWARE


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Designer Chromosome and the Additional Genes

Electronmicrograph of DNA


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DESIGNER BACTERIA PRODUCING A VARIETY OF ANTIBIOTICS

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

Documents

MICR3003 Lecture 2 Designer BacteriaDr J M Pemberton 2003