bacterial transformation

2007-2008

Bacterial Transformation

Lab 6A

Our Goals1. Insert foreign gene into plasmid2. Insert recombinant plasmid into

bacteria3. Grow transformed bacteria

Questions We Need Answered1. Did the bacteria take up the plasmid?2. 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

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

Engineered Plasmids We will be using pBlue

Lac-Z gene

Ampicillin Resistancegene

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

Lac-Z gene

Ampicillin Resistancegene

Restriction site

We will be using 3 types of agar1. LB2. LB + amp3. LB + amp + X gal

Each Group Will Run Six Plates

LB- plasmid

LB/amp- plasmid

LB/am/X-gal

- plasmid

LB+ plasmid

LB/amp+ 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

LB + amp

only transformed bacteria grow

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?

What if your – shows unexpected growth?

Can you tell by looking at + which ones are recombinant?

LB/amp Plates

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

recombinantplasmid

ampresistance

“broken”LacZ gene

insertedgeneof interest

LB + amp + X gal

Blue vs. White on X-gal PlateBacteria take up plasmidFunctional LacZ geneX gal is being digestedBacteria make blue color

Bacteria take up recombinant plasmidNon-functional LacZ geneX gal is NOT being digestedBacteria 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!

AnyQuestions?