Bacterial Transformation Lab 6A 2007-2008

Our Goals Insert foreign gene into plasmid Insert recombinant plasmid into bacteria Grow transformed bacteria

Questions We Need Answered Did the bacteria take up the plasmid? If so, did the gene of interest get inserted into the plasmid? Analyzing our plates at the end of the lab will answer these questions and tell us if we achieved our goals

What must you know to understand this lab? Essential Knowledge What must you know to understand this lab?

Antibiotic Resistance Resistant bacteria can survive in the presence of antibiotics We will be using ampicillin Our plasmid is resistant to ampicillin

Lac-Z gene Lactose = glucose + galactose Lactose is broken down by the Lac-Z gene Lac-Z will also digest similar compounds

X gal X gal is a compound that is similar to lactose X gal turns blue when it is digested by the Lac-Z gene

Ampicillin Resistance Engineered Plasmids We will be using pBlue Lac-Z gene Ampicillin Resistance gene

Restriction Enzymes Our plasmid DNA and our foreign DNA arrive pre-cut by the same restriction enzyme The restriction enzyme made a single cut in the plasmid The restriction site is within the lac-Z gene

Ampicillin Resistance Restriction site Lac-Z gene Ampicillin Resistance gene

We will be using 3 types of agar LB + amp LB + amp + X gal

Each Group Will Run Six Plates LB - plasmid LB + plasmid LB/amp - plasmid LB/amp + plasmid LB/am/X-gal - plasmid LB/am/X-gal + plasmid

How will we know if we are successful?

LB Plates There is nothing in the LB agar to interfere with growth. Growth on both the + and – plates indicates the bacteria are alive and well. A “lawn” is too many colonies to count - plasmid + plasmid

What if nothing grows on either LB plate?

LB/amp Plates Only bacteria that have taken up the plasmid will grow on the plates with ampicillin How do you know?

If the plasmid is inserted into the bacteria, it will become antibiotic resistant and will grow on the ampicillin plate only transformed bacteria grow LB + amp

LB/amp Plates No growth on – confirms ampicillin is working because bacteria without the plasmid are not resistant and die Bacteria growing on + contain the plasmid and are amp resistant - plasmid + plasmid

LB/amp Plates Would you expect isolated colonies or a lawn on the + ? Why?

LB/amp Plates What if your – shows unexpected growth? Can you tell by looking at + which ones are recombinant?

LB/amp/X-gal Color of colonies on X-gal plate will tell you if gene of interest has been inserted into the plasmid or not Blue colonies indicate Lac Z gene is digesting X-gal White colonies indicate Lac Z gene in not functioning

LB/amp/X-gal If ends of lac Z gene come back together and are resealed by DNA ligase during the transformation process, the Lac Z gene will be functional and will turn X gal blue.

LB/amp/X-gal If the gene of interest is successfully inserted between the free ends of the plasmid, the Lac Z gene will be “broken”. If the Lac Z gene is interrupted, X-gal will not be digested and the colonies will remain white

If the foreign DNA is inserted into the plasmid, The Lac-Z gene will be interrupted and bacteria will stay white recombinant plasmid amp resistance “broken” LacZ gene inserted gene of interest LB + amp + X gal

Blue vs. White on X-gal Plate Bacteria take up plasmid Functional LacZ gene X gal is being digested Bacteria make blue color Bacteria take up recombinant plasmid Non-functional LacZ gene X gal is NOT being digested Bacteria stay white color

What if you just see blue colonies on + ? Should you ever see blue colonies on - ? Should you see isolated colonies or a lawn on the X gal plate? Why? - plasmid + plasmid

What do we really want to see?

White Colonies on X-gal ! LB + amp + X gal SUCCESS!

Any Questions?