1. Bacterial Physiology (Micr430)
Lecture 13
Bacterial Genomics and Functional Genomics
(from various sources)

2. Genome and Proteome
Genome: The complete genetic information carried by an organism
Proteome: Encompasses all the expressed proteins of an organism (protein array)
Gene array: an orderly array of sequence tags each specific for one gene of a genome; (other names: gene chip, DNA microchip)

3. Genomics and Functional Genomics
Genomics: The study of all of the nucleotide sequences, including structural genes, regulatory sequences, and noncoding DNA segments, in the chromosomes of an organism
Functional genomics: the branch of genomics that determines the biological function of the genes and their products

4. Protein
array

5. Gene Array or DNA microaray
There are two main applications
Identification of gene or mutation
Determination of expression level of genes
There are two formats:
cDNA (500 – 5000 bp) molecules are spotted on the chip
Array of oligonucleotides are synthesized and immobilized on the chip

6. Gene Array or DNA microarray
1) Probe (cDNA/oligo with known identity)
2) Chip fabrication (Putting probes on the chip)
3) Target (fluorecently labeled sample)
4) Assay
5) Readout
6) Informatics

9. DNA microarray applications
Gene discovery
Study of gene functions and expression
Study relationship of genes
Disease diagnostics – infectious diseases

10. Study of gene function
Gene replacement (knock-out): also known as reverse genetics. The purpose is to remove (knock-out) most of one gene and see what happens to the phenotype of the organism. Suicide vector is used
Transposon mutagenesis. Similar to above but is more random disruption of genes.

12. Study of gene function
Antisense RNA expression. Random cloning and expression of short pieces of genomic DNA on a plasmid in an microorganism to elucidate the function of the genes
Essential gene: a gene whose function is absolutely required for the growth and survival of a cell.

13. Conditional Antisense Inhibition of Protein Synthesis
Inducible promoter
Normal cell
Antisense cell
Plasmid DNA No
protein
Protein
Antisense RNA X
mRNA
mRNA
DNA
DNA

14. Shotgun Antisense Expression Determines Essentiality of Genes
Pathogen genome
Millions of random DNA fragments
Non essential gene
blocked by antisense
Essential gene
No cell growth
mRNA
Essential Protein
DNA

15. Ultra-Rapid Functional Genomics
Identify >100 essential gene drug targets per month
Antisense
(+ inducer)
No antisense
(- inducer)

16. Traditional Tools
Reporter gene: A gene whose product can be easily assayed
Transcriptional gene fusions
Translational gene fusions
PCR
DNA Mobility Shift

18. PCR

19. Gel Shift
Assay