Bacterial Transformation Lab

Introduction

Biotechnology refers to technology used to manipulate DNA. The procedures are often referred to as genetic engineering.

DNA is the genetic material of all living organisms and all organisms use the same genetic code. Genes from one kind of organism can be transcribed and translated when put into another kind of organism.

For example, human and other genes are routinely put into bacteria in order to synthesize products for medical treatment and commercial use. Human insulin, human growth hormone, and vaccines are produced by bacteria.

Recombinant DNA refers to DNA from two different sources. Individuals that receive genes from other species are transgenic.

Vectors

Vectors are DNA used to transfer genes into a host cell.

Plasmids can be used as vectors to transform bacteria. The host bacterium takes up the plasmid, which includes the foreign gene.

Marker genes can be used to determine if the gene has been taken up. Marker genes must have some distinguishable characteristic. For example if you put a gene that enables an ampicillin resistance on the same vector as the same vector as the gene for human insulin production, then any bacteria that grow on an ampicillin plate will be able to produce insulin.

Gene Regulation

The bacterium Escherichia coli has a plasmid that contains genes that enable it to digest the sugar arabinose. The plasmid also contains elements which allow the gene to be turned on or turned off. Arabinose is not normally found in the environment of E. coli, so it is beneficial for the genes to be turned off. This prevents the synthesis of proteins that are not needed. When arabinose is present, genes that produce proteins that digest arabinose are switched on and they produce their enzymes. A simplified diagram of the plasmid is below.

pGLO Plasmid

A plasmid has been constructed in which some of the genes needed for digesting arabinose have been replaced by a gene that codes for green fluorescent protein. The protein glows green when viewed under UV light. The plasmid is called pGLO.

The pGLO plasmid also has a gene that gives the bacterium resistance to the antibiotic ampicillin. Normally ampicillin kills bacteria, but those with this gene can survive. A simplified diagram of the pGLO plasimd is below.

We will put the pGLO plasmid in E. coli. We know that the bacteria have taken up the plasmid if 1) they glow green under UV light and 2) if they survive when grown on a medium that contains ampicillin.

Materials Needed

The following equipment will be needed for each group:

1 E. coli starter plate

4 agar plates:

        1 LB plate

        2 LB/amp plates

        1 LB/amp/ara plate

4 microtubules (ignore the colors of the tubules):

        1 microtubule containing transformation solution

        1 microtubule containing LB broth

        1 empty microtube labeled + (or + DNA)

        1 empty microtube labeled - (or - DNA)

1 foam microtube holder

1 container with ice water

1 package of. inoculation loops

5 pipettes

Procedure

Add Transformation Solution (CaCl₂)

Using a sterile pipette, transfer 250 ul of transformation solution (T.S.) to the tube labeled + DNA and another 250 ul to the tube labeled - DNA. The 250 ul line is the one below the 0.5 ml line. It is the third line from the tip.

Place the tubes back in the foam microtube holder and then float all four of the tubes in a container of ice water for 2 minutes.

Add Bacteria

When transferring bacteria to or from an agar plate, use the technique shown below. Organisms are transferred by using a sterile loop and reaching in from the side while keeping the plate covered as much as possible. This technique minimizes the risk of contamination from above.

Use a sterile loop to pick up several colonies of bacteria from the starter plate. This can be done by dragging the loop across the plate so that it lightly scrapes the colonies off the surface. Transfer the bacteria to the + DNA tube by spinning the loop rapidly after it is immersed in the liquid.

Repeat this procedure by transferring several colonies of bacteria to the - DNA tube.

Add DNA

Your instructor will provide you with a bottle containing plasmid DNA. Immerse a sterile loop into the bottle containing plasmid DNA. When the center of the loop is coated with a soap-like film, transfer it the + DNA microtube (see photograph below). Use a new sterile loop to transfer a second loopful of plasmid DNA into the same (+ DNA) microtube. The - DNA microtube will not receive any plasmid DNA.

Float the two tubes in their foam holder in ice water for 10 minutes.

Summary

The + DNA tube contains bacteria and DNA from another source. The - DNA tube contains only bacteria.

While Waiting...

While waiting for the tubes to cool, use a marker to label your agar plates with a letter that indicates the type of bacteria that they will receive.

Plate Label Comments

LB/amp T this plate will receive transformed bacteria

LB/amp/ara T this plate will receive transformed bacteria

LB/amp N this plate will receive normal, untransformed bacteria

LB N this plate will receive normal, untransformed bacteria

Heat Shock

Bring your container of ice water and the microtubes to the 42 degree water bath.

Transfer the foam microtube holder containing the + and - tubes to a 42 degree C water bath for exactly 50 seconds then return the tubes to ice and water. It is important that this heat shock last for exactly 50 seconds. In order to insure that the time is exactly correct, have one person time the procedure while another transfers the tubes to the water bath and returns them when 50 seconds has elapsed.

Allow the tubes to remain in the ice water for 2 minutes.

Remove the foam microtube holder containing the microtubes from the ice water and place it on your bench top. Add 250 ul of LB broth to each of the bacterial cultures (the + tube and the - tube) with a sterile pipette.

Allow the tubes to stand at room temperature for 10 minutes.

Transfer Bacteria to the Culture Plates

Mix the two tubes by tapping them with the fingernail of your index finger.

Using a sterile pipette, transfer 100 ul of the solution from the + microtubule to the surface of the agar in one of the plates labeled T. The 100 ul line is the second one from the tip on the pipette. Transfer another 100 ul to the other plate labeled T.

Using a different sterile pipette, transfer 100 ul of solution from the - microtubule to the surface of the agar in one of the plates labeled N. Transfer another 100 ul to the other plate labeled N.

Spread the mixture over the entire surface of the agar in one of the plates using a sterile loop. Use different sterile loops to spread the mixtures in the other plates.

Incubate the Plates

Turn the plates over so that the agar is up and the cover is on the bottom side. Stack them one on top of the other and then tape them together. Write your name on the tape.

Your instructor will transfer your plates to the 37 degree incubator in the Microbiology Laboratory. They will remain in the incubator for 24 to 48 hours.