Microbial Genomes and techniques for studying them. How do we figure out how Listeria tolerates acid?

Remember: DNA – RNA – Protein Enzymes + food = new cells Acclimation to stress:

Learning goals Compare and contrast the techniques used to understand microbial genomes and their functions PCR Sanger sequencing Next gen sequencing transcriptomics Explain how they can be used to discover mechanisms for acclimation to stress

How to determine what is in a genome PCR Sanger sequencing Next – generation sequencing How to determine what is in a genome

Useful properties of DNA for biotechnology DNA melts at high temperatures Single strands Melting Tm  85.0° Double strand 1.2 1.0 0.8 72 76 80 84 88 92 96 °C A260 Sequences are complementary CCGGTA C G GGCCAT G C T A A T G

The polymerase chain reaction Given these ingredients – how can we copy DNA in a tube, w/out cells? Ingredients: Taq DNA polymerase dNTPs Buffer for enzyme 2 primers – one for each strand Template: Genomic DNA that you are copying Thermalcycler: 55oC 94oC 72oC

At the end of a PCR reaction, is all of the DNA in the tube the same size? Why or why not?

Sanger sequencing Ingredients PCR product of the region you want to sequence Forward OR Reverse primer that you used for PCR ddNTPs dNTPs Taq DNA polymerase Appropriate buffer for Taq Water Thermal cycler

Sanger sequencing demo: PCR products CCGGTA C G A T T A C CCGGTA C G A T T A C GGCCAT G C T A A T G GGCCAT G C T A A T G CCGGTA C G A T T A C CCGGTA C G A T T A C GGCCAT G C T A A T G GGCCAT G C T A A T G CCGGTA C G A T T A C CCGGTA C G A T T A C GGCCAT G C T A A T G GGCCAT G C T A A T G

Sanger sequencing demo CCGGTA C G A T T A C GGCCAT G C T A A T G CCGGTA C G A T T A C GGCCAT G C T A A T G CCGGTA C G A T T A C GGCCAT G C T A A T G CCGGTA C G A T T A C GGCCAT G C T A A T G CCGGTA C G A T T A C GGCCAT G C T A A T G GGCCAT G C T A A T G CCGGTA C G A T T A C

Sanger sequencing demo For brevity during this demo, X = normal nucleotide When a ddNTP is incorporated, we will mark it with the nucleotide symbol (A, T, C, or G) and * CCGGTA C G A T T A C CCGGTA C G A T T A C GGCCAT GGCCAT CCGGTA C G A T T A C CCGGTA C G A T T A C GGCCAT GGCCAT CCGGTA C G A T T A C CCGGTA C G A T T A C GGCCAT GGCCAT

Sanger sequencing demo CCGGTA C G A T T A C 1000 GGCCAT G* G* CCGGTA C G A T T A C 1000 GGCCAT G C* C* CCGGTA C G A T T A C 1000 GGCCAT G C T* T* CCGGTA C G A T T A C 1000 GGCCAT G C T A* A* CCGGTA C G A T T A C 1000 GGCCAT G C T A A T A*

Sanger sequencing demo GGCCAT G C T A A T* A* X 1000 GGCCAT G C T A* A* X 1000 GGCCAT G C T* T* X 1000 Tiny tube with charged buffer GGCCAT G C* C* X 1000 GGCCAT G* G* X 1000

Sanger sequencing demo GGCCAT G C T A A T* A* X 1000 GGCCAT G C T A* A* X 1000 GGCCAT G C T* T* X 1000 Tiny tube with charged buffer GGCCAT G C* C* X 1000 GGCCAT G* G* X 1000 Machine records quantity of fluorescence/color/time

Sanger sequencing demo GGCCAT G C T A A T* A* X 1000 GGCCAT G C T A* A* X 1000 GGCCAT G C T* T* X 1000 Tiny tube with charged buffer GGCCAT G C* C* X 1000 GGCCAT G* G* X 1000 Machine records quantity of fluorescence/color/time

Sanger sequencing demo GGCCAT G C T A A T* A* X 1000 GGCCAT G C T A* A* X 1000 GGCCAT G C T* T* X 1000 Tiny tube with charged buffer GGCCAT G C* C* X 1000 GGCCAT G* G* X 1000 Machine records quantity of fluorescence/color/time This reaction has two different sequences in it. How can you tell?

Sanger Sequencing demo study Q’s Why do have to start with many copies of the gene? (hint: can the new strand be used as a template for entire molecules?) Do you think the ratio of dNTPs to ddNTPs is important? What if more than half of the nucleotide pool were ddNTPs? 1/1000 of the nucleotide pool? What would happen if you put both forward and reverse primers in the reaction? Get replication? Pure sequence? Think about what goes through the tube….

In each sanger reaction, we can sequence basically one gene, more or less How many sanger reactions would we need/genome? E. coli chromosome 4288 protein coding genes, 88% of genome 1% of genome is tRNAs and rRNAs 10% of genome = regulatory sequences 0.5% repetitive non-coding sequences

Next gen sequencing is faster than Sanger Ex. Illumina

Sequencing lots of genes with Sanger PCR amplify the gene with correct primers Purify PCR product Sequencing reaction Interpret chromatogram Gene 1 PCR amplify the gene with correct primers Purify PCR product Sequencing reaction Interpret chromatogram Gene 2 PCR amplify the gene with correct primers Purify PCR product Sequencing reaction Interpret chromatogram Gene 3 PCR amplify the gene with correct primers Purify PCR product Sequencing reaction Interpret chromatogram Gene 5700

Why next gen is faster than sanger PCR

Next-generation sequencing is high throughput Illumina

Next-generation sequencing is high throughput Illumina

Next-generation sequencing is high throughput Illumina

Next-generation sequencing is high throughput Illumina

Next-generation sequencing is high throughput Illumina PCR

Next-generation sequencing is high throughput Illumina Sanger in each cluster, Colors change in each spot as bases are added =fluor, over time AND space PCR

Next-generation sequencing is high throughput Illumina Sanger in each cluster, Colors change in each spot as bases are added =fluor, over time AND space PCR

http://www.illumina.com/technology/next-generation-sequencing/paired-end-sequencing_assay.html

How do we go from DNA sequence to knowing genes? What do we know about genes? Start and stop codons, protein length, promoters, evolution and similar functitons How do we go from DNA sequence to knowing genes?

Example model of cell from genome seq

Gene expression links sequence to phenotype Which genes are used by the cell in different conditions? Ex. Acid stress vs no acid vs a different acid

Transcriptomics experimental design

Melting and hybridization and probes Remember this? Single strands Melting Tm  85.0° Double strand 1.2 1.0 0.8 72 76 80 84 88 92 96 °C A260 Sequences are complementary CCGGTA C G GGCCAT G C T A A T G

Transcriptomics – quantifying different mRNAs Probes: short DNA strands attached to a slide in known arrangement Target: the cDNA that can hybridize with a particular probe

End product: Transcriptomics

We can also deduce regulation Bacteria have operons

Regulons

Gene regulatory networks