Modeling Transformation

Purpose: To understand the process and rationale for bacterial transformation.

Background:

Genetic transformation occurs when a cell takes up and expresses a new piece of DNA in order to change its traits. In addition to one large, circular chromosome, many bacteria naturally contain one or more tiny circular pieces of DNA called plasmids. Plasmid DNA contains genes for traits that may be helpful for a bacterium to survive under certain environmental conditions. In nature, bacteria can transfer plasmids back and forth, allowing them to share these beneficial genes and adapt to new environments.

Materials: Yarn, sequins, black Velcro, green Velcro, pink Velcro, water bottle, pipe cleaner, plastic cup, adhesive dots, labels

Procedures:

1. Construct a pGLO plasmid by fastening both the green (GFP) and pink (araC) Velcro pieces to the black Velcro piece (plasmid DNA).

2. Build a bacterial cell by sticking the adhesive dots (negative charges) on the outside of the water bottle (cell membrane).

3. Lace the pipe cleaner through the hole at the bottom of the water bottle to represent the flagellum.

4. Place the blue yarn (chromosomal DNA) and red sequins (ribosomes) inside the water bottle (cell). Replace the cap.

5. Draw and label a diagram of your bacterial cell under Observations.

Transformation

6. Add the transformation solution (CaCl2) to your micro-tube.

7. Inoculate the micro-tube with your bacterium.

***Remove the adhesive dots from the water bottle.

8. Incubate the micro-tube on ice. ***Remove the bottle cap.

9. Heat-shock the micro-tube for 50 seconds in the 42°C water bath.

***Push the Velcro plasmid into the water bottle.

10. Return the micro-tube to ice. ***Replace the bottle cap.

11. At room temperature, add nutrient broth (LB) to the micro-tube.

Observations:

FIGURE 1: Labeled diagram of bacterial cell model

Data:

TABLE 1

Transformation Step / Purpose
Addition of CaCl2
1st incubation on ice
Heat shock
2nd incubation on ice
Addition of nutrient broth

Hypothesis: For each plate, make a prediction about what you expect to see.

Plate / Plasmid (pGLO) / Grow? / Glow?
LB / - / yes / no / yes / no
LB/Amp / - / yes / no / yes / no
LB/Amp / + / yes / no / yes / no
LB/Amp/Arabinose / + / yes / no / yes / no

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