Bacterial Transformations and PCR (Part I of II) Exercises 5 and 6 Bacterial Transformations and PCR (Part I of II)

Learning Outcomes Describe the structure and function of plasmids Summarize the steps and requirements for bacterial transformations Perform bacterial transformation Isolate genomic DNA from check swab Summarize the steps and requirements for PCR Prepare a PCR sample

Procedures PLASMID STUFF 5.1: Digest plasmid with restriction enzymes: SKIP 5.2: Transform Bacteria with pGLO plasmid 5.3: Bacterial Growth and Selection GENOMIC STUFF 5.4: Isolate your DNA 5.5: Prepare your DNA for PCR

Plasmids Some bacteria have smaller rings of DNA called plasmids Plasmids replicate separately from the bacterial chromosome Bacterium with a plasmid plasmid Engineered plasmid Antibiotic resistance gene

Transforming Bacteria (5.2) Bacteria can be made ‘chemically competent’… meaning they will take up plasmids from the environment Begin by treating E. coli with a high concentration of calcium ions Makes their phospholipid bilayer ‘leaky’’ Are now ‘chemically competent’ We will provide you with chemically competent bacteria! We will provide you with the pGLO plasmid

We will give you chemically competent E. coli We will give you the pGLO plasmid You will transform bacteria with the pGLO plasmid: Heat shock followed by recovery Only a few cells will successfully incorporate a plasmid.

Your plasmid: pGLO Components of your pGLO plasmid ori (origin for DNA replication) bla (ampicillin resistance) Multiple Cloning Site (MCS) GFP gene araC gene bla gene: codes for the enzyme beta-lactamase: gives transformed bacteria resistance to the beta-lactam family of antibiotics (such as ampicillin) A bacterium that incorporates the pGLO plasmid (is transformed) has what ‘new’ features?

GFP Gene GPF gene codes for the green fluorescent protein. It is composed of 238 amino acids and fluoresces bright green when exposed UV light. Although many other marine organisms have similar green fluorescent proteins, GFP traditionally refers to the protein isolated from the jellyfish Aequorea victoria No UV UV

araC protein (produced by the araC gene) In the absence of the sugar arabinose, the araC protein prevents transcription of the GFP gene. In the presence of arabinose, the araC protein no longer prevents transcription of the GFP gene. The result…. RNA polymerase transcribes the GFP gene and the fluorescent protein is produced.

Bacteria Require a growth medium (LB media) 10 g tryptone (a.a.) 5 g yeast extract (nutrients) 10 g NaCl (osmotic balance) Elective growth compounds may also be added to the media, such as antibiotics. Pour the media into a petri dish Individual microorganisms added using a technique known as "streaking” Loop

Bacterial lawn vs colonies A bacterial lawn: forms when all the individual colonies merge to form a field or mat of bacteria. A colony of bacteria: all bacteria in the colony are descendants of a single mother cell (bacteria all genetically identical in a colony)

5.3 Bacterial Growth + LB +LB +LB + AMP +AMP – AMP + ARA -ARA – ARA You will be given three different plates: + LB + AMP + ARA +LB +AMP -ARA +LB – AMP – ARA 1 2 3 On which plate(s) do you expect to see a lawn? On which plates(s) do you expect to see colonies? On which plate(s) do you expect transformed bacteria to grow? (genotype +plasmid) On which plate(s) do you expect GFP to be produced? Will transformed bacteria always fluoresce? On which plate(s) do you expect the bacteria to fluoresce? (phenotype +fluoresce)

WHAT DETERMINES PHENOTYPE? 1) Only those bacteria that have been transformed will survive on plates with ampicillin. 2) Arabinose allows for the transcription of the gene that codes for GFP. 3) Fluoresce ONLY in UV light With the right genotype AND environmental conditions….you get glowing bacteria! Genotype + Environment = PHENOTYPE

Genomic DNA Isolation 5.4.3: Collect Cells (swab) 5.4.5: Disrupt Cell membranes (Genomic Lysis Buffer) 5.4.9: Spin to discard cell debris, DNA Binds to matrix 5.4.12 and 5.5.14: Removing impurities (Wash buffer) 5.4.16: DNA released from matrix, collected

Isolate Human DNA from Buccal Samples You will isolate all 46 of your chromosomes during lab from cheek cells using a kit (5.4) However, you will study only one small region (locus) on chromosome 1 (D1S80 locus). D1S80 locus located on distal end of short arm ‘p’ (1p36) What is at this locus? Chromosome 1 is the designation for the largest human chromosome. Humans have two copies of chromosome 1, as they do with all of the autosomes, which are the non-sex chromosomes. Chromosome 1 spans about 249 million nucleotide base pairs, which are the basic units of information for DNA.[2] It represents about 9% of the total DNA in human cells.[3] Identifying genes on each chromosome is an active area of genetic research. Chromosome 1 is currently thought to have 4,316 genes, exceeding previous predictions based on its size.[2] It was the last completed chromosome, sequenced two decades after the beginning of the Human Genome Project.

The Human Genome https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1186966/ A variable number tandem repeat (or VNTR) is a location in a genome where a short nucleotide sequence is organized as a tandem repeat. These can be found on many chromosomes, and often show variations in length (number of repeats) among individuals. Each variant acts as an inherited allele, allowing them to be used for personal or parental identification. Their analysis is useful in genetics and biology research, forensics, and DNA fingerprinting.

D1S80 Minisatellite or VNTR’s (variable number tandem repeats) 16 bp repeat unit [GAGGACCACCGGCAAG] 16 bp repeat unit repeats 20 times Sam 16 bp repeat unit repeats 15 times 16 bp repeat unit repeats 20 times Beth 16 bp repeat unit repeats 20 times

D1S80 28 known alleles in human population 14 - 41 repeats 16(r) +146 = 370 to 802 bp Individuals can be homozygous or heterozygous 80% of population is heterozygous

D1S80 How will you figure out the size of your alleles and number of repeats? How will you figure out whether you are homozygous or heterozygous? PCR and Gel Electrophoresis!

The Steps of Polymerase Chain Reaction 1. Denaturation – heating the mixture to 95°C separates the two strands of the DNA. 2. D1S80 Primer annealing – cooling the mixture allows the D1S80 primers to bond, or anneal, to complementary sections of single-stranded target DNA flanking D1S80 locus. 3. Extension – heating the mixture to 72°C causes the Taq polymerase to synthesize the complementary DNA strand from the dNTPs, starting at the primer. Steps 1–3 are continually repeated to yield the necessary number of copies. http://highered.mheducation.com/olc/dl/120078/micro15.swf

Polymerase Chain Reaction (PCR): making copies of targeted regions of DNA Ingredients Your DNA (all 46 chromosomes). Each student will make their own tube. D1S80 Primers MasterMix: Taq polymerase, MgCl2, DNA building-blocks, deoxyribonucleoside triphosphates (dNTPs) Thermus aquaticus dGTP Taq polymerase

Forward: 5'-GAAACTGGCCTCCAAACACTGCCCGCCG-3' Forward and Reverse D1S80 primers correspond to the sequences on either side of the D1S80 locus to be copied! We buy them for you! Forward: 5'-GAAACTGGCCTCCAAACACTGCCCGCCG-3' Reverse: 5'-GTCTTGTTGGAGATGCACGTGCCCCTTGC-3' DNA polymerase D1S80 LOCUS DNA polymerase

Positive control (prep staff) 5.5: PCR Setup Tube letter Sample NAME Master mix DNA template Primer Mix D1S80 Total Volume A Student #1 genomic DNA 12.5 ul 10.0 ul D1S80: 2.5 ul 25.0 ul B Student #2 genomic DNA C Student #3 genomic DNA D Student #4 genomic DNA D1S80: 2.5 u E Negative control 10.0 ul dH2O Positive control (prep staff) Master Mix: Taq polymerase, MgCl2, dNTPs and buffer Remember to keep your samples on ice and ALWAYS use a new sterile pipette tip with aerosol-barrier (labeled for PCR-Only) for each transfer to avoid contaminating the stock reagents as well as your samples! How is the negative control different? What is its purpose? How is the positive control different? What is its purpose?

Place the tubes in the PCR machine Thermocycler

What will be amplified. All 46 chromosomes What will be amplified? All 46 chromosomes? The entire length of Chromosome 1? A small segment or locus on Chromosome 1?