Microbial genetics introduction

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  • Created by: lridgeway
  • Created on: 03-11-20 12:01

Why study microbial genetics?

Geneticist/biochemist perspective: useful model systems for understanding fundamental processes common to all forms of life. Many discoveries have been found in microbes e.g. genetic code, process of transcription and translation, metabolic pathways and many more.

Much of our undertstanding of gene structure and function has derived from genetic analysis of model bacterial and bacteriophage systems.

Microbiologists perspective: understanding ane exploiting their biology. Ecology - ubiquitous, occupy niches, essential for N2 fixation and geochemical cycles. Cell biology is complex and dynamic. Pathogenicity is useful for treatment and control of disease. Useful in biotechnology for antibiotic and new chemical production. 

All areas of modern microbiology require knowledge of microbial genetics. 

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Advantages of using microbes for genetics

  • Reproduce rapidly
  • Simple to maintain and cultivate 
  • Large numbers of identical individual cells can be produced in a short amount of time (inportant for mutant screens) 
  • Human genome sequence still expensive in comparison to bacteria amd can gain useful information from both 
  • Populations large enough to contain spontaneous mutants (freq can then be increased using mutagenic agents). Selection techniques can then allow detection of one muatant within large populations of cells. 

Advantages of bacteria for studying genetics

  • Bacteria have haploid genomes for phenotype of mutation seen immediately 
  • Relatively small genome 
  • Genetic manipulation straightforward (depending on organism) 
  • We can amke strains carrying desired combinations of mutations with relative ease
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Creating first synthetic bacterium

Wanted to do this to make it as efficient as possible to grow biofuels using bacteria. 

Steps are: 

  • Design the bactria computationally 
  • Synthesise fragments of DNA
  • Assemble and contruct genome
  • Clone into a yeast for proagation 
  • Isolate and transplant into Mycoplasma capricolum
  • Restriction enzyme was included in the design to degrade the native chromosome which completes the process 
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Forward genetics

Biological function (phenotype) to gene 

Phenotypic screen for desired mutants and then have biochemical characterisation of the mutants. Grow up sample of mutants. Genetic analysis (sequencing/complementation test etc) and gene isolation occur in order to study the gene product. 

Advan: Emphasis on desired phenotype. Can find mutants with defects in essential genes

Disadvan: Slow. May be impossible to find all the genes in a species for a specific phenotype or to find the specific gene is lots of mutations are present. 

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Reverse genetics

From gene to phenotype. Done by altering gene sequence or inbiting expression of the gene. 

Only become possible due to the advancements in molecular genetics. Harder than forward genetics as you have to screen without knowing a phenotype. 

Best way to go about it is to look at microbial genenomes that have already been sequenced and see if your gene of interest has homolgy to a known gene that has been charcterised as this may give an idea of what phenotype to expect. Then you want to mutate the gene in vitro and substitue the mutated allele for the WT one in genome. Finally you want to determine the phenotype of the mutanat strain (most difficult part). 

A common way to find a phenotype is using a BiOLOG phenotype array. This is 20x96 well plates all with defined media (cab be altered) such as different carbon sources or antibiotics etc. 

Clear wells on a plate means that well isnt used as not all bacteria use the same sources for things. In a carbon source example a purple well is caused by NADH production. Yellow shows weak growth and a change from purple to yellow when comparing WT and a mutant usually indicates the changed genes phenotype. 

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Uses of mutants

  • Help to idetify genes involved in a particular function - e.g transposon insertions in genes (a good library has at least one transposon per gene) 
  • Mutant phenotypes can be informative - e.g pathway blockages result in intermediate accumulation
  • Undertsanding metabolic regulation - e.g mutant transcription factors lead to gene upregulation 
  • Identifying the source of action of an antibiotic -e.g a mutant with changes the site of binding for the antibiotic can survive if plated on the antibiotic suggesting that is the site of action of the antibiotic 
  • Find conditional lethal mutants - e.g find genes that are essential for viability depending on the conditions for example a temperature senstive mutant where mutation is only expressed above a certain temp. Below it would behave like a WT. 
  • Having a mutant can help to clone genes - add a WT gene to a mutant cell can confer complementation and restore the WT phenotype if the WT gene inserted matches the mutation in the cell. This can be done if the WT phenotype is selectable and a gene library of plasmids is transformed into mutant cells. The one that confers sensitivity can be sequenced as this is where the mutation in the cell is. 
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Types of mutation

It is a heritable change in DNA sequence. 

Point mutations change 1 bp (aka SNPS). If they are between two purines or two pyrimidines they are transitions but if they are between a purine and a pyrimidine they are known as transversions. 

Larger mutations:

  • Insertion of a section of DNA into a chromosome 
  • Deletion of a portion of chromosome
  • Inversion - flipping a portion of chromosome 
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