Genes are pieces of DNA that code for certain traits. By definition, genetic transformation is when an organism's traits are changed by way of an inserted, manipulated gene. Agriculture, bioremediation, and medicine, are all fields for which genetic transformation is applicable because plants can be modified for better harvest, bacteria can help clean oil spills, or transform a sick person's cells to healthy ones. For the genetic transformation in this lab, we will transfer a gene to the circular pieces of DNA in bacteria called plasmids. These plasmids contain beneficial genes for the bacteria's survival, so when a new environment is introduced, it can adjust by transferring DNA with its environment. The gene desired for expression in the bacteria is the Green Fluorescent Protein, or GFP, found in bioluminescent sea jellies.
Objective:
The objectives are to genetically transform bacteria to express the GFP gene. If we are successful, the bacteria should glow in the dark.
Procedure:
- Label one test tube +pGLO and another =pGLO, label with table number
- Open the tubes and transfer 250 microliters of CaCl2 (100-1000 microliters)
- Place the tubes in ice
- Pick up a visible clump of bacteria with a sterile loop, immerse the loop in the transformation solution in the +pGLO tube, no chunks!, Place the tube in ice. Repeat for -pGLO
- Add 10 microliters of plasmid DNA directly to liquid in +pGLO tube only, flick tube to mix, close tube and place in ice. DO NOT ADD PLASMID TO -pGLO tube
- incubate the tubes on ice for 10 minutes, make contact with the ice
- Labels: LB/amp plate: pGLO; LB/amp/ara plate: +pGLO; LB/amp plate: -pGLO; LB plate: -pGLO
- place the + and - pGLO tubes into the water bath at 42 degrees Celsius for 50 secs. Place tubes back on ice after. Incubate tubes for 2 minutes.
- Add 250 microliters of LB nutrient broth to the tube and reclose it, repeat for each tube, incubate for 10 minutes
- Tap closed tubes with finger to mix. Pipet 100 microliters of transformation and control suspensions onto the appropriate plates.
- Use a new sterile loop for each plate. Spread the suspensions evenly around the surface of the agar by skating across back and forth.
- Stack up the plates, tape them together, put table and period, place in incubator upside down!
- Clean up and return all supplies
Due to the colder environment or the insufficient plasmid, the bacteria failed to glow. The bacteria grew slightly.
