Molecular Biology
Abstract
The bacterium used in this lab, Escherichia coli (or E. coli) is an ideal organism for the molecular geneticist to manipulate. It can easily be grown in suspension culture in a nutrient medium such as Luria broth, or in a petri dish of Luria broth mixed with agar (LB agar) or nutrient agar.
Genes can be transferred between bacterial in three ways: conjugation, transduction, or transformation. Bacterial transformation involves transfer of genetic information into a cell by direct uptake of the DNA. During gene transfer, the uptake and expression of foreign DNA by a recipient bacterium can result in conferring a particular trait to a recipient lacking that trait. Transformation can occur naturally but the incidence is extremely low and is limited to relatively few bacterial strains.
Plasmids can transfer genes that occur naturally within them, or plasmids can act as carriers for introducing foreign DNA from other bacteria plasmids, or even eukaryotes into recipient bacterial cells.
In this lab, the LB- and LB+ plates had a lawn of growth, the most growth out of all the plates. The LB/amp+ plate also showed some bacterial growth, but it was very little. The LB/amp- plate was the only plate that had no observed bacterial growth. Transformation efficiency might be affected by the picking up of enough cells, the time of cold and heat shocking, not re-suspending, and not using aseptic technique. The lawn on growth observed in the LB- and LB+ plates are due to the absence of ampicillin. The reason why the LB/amp+ plate showed some growth was because of the resistant plasmids. Since there were no plasmids to resist the ampicillin in the LB/amp- plate, there was no growth.
Introduction
The bacterium used in this lab, Escherichia coli (or E. coli) is an ideal organism for the molecular geneticist to manipulate and has been used extensively in recombinant DNA research. It is a common inhabitant of the human colon and can easily be grown in suspension culture in a nutrient medium such as Luria broth, or in a petri dish of Luria broth mixed with agar (LB agar) or nutrient agar.
E. coli contains about five million DNA base pairs in its singular circular chromosome. E. coli may also contain small circular DNA molecules called plasmids, which also carry genetic information. The plasmids are extrachromosomal; they exist separately from the chromosome. Some plasmids replicate only when the bacterial chromosome replicates, and often occur in as many as 10 to 200 copies within a single bacterial cell. Certain plasmids, called R plasmids, carry genes for resistance to antibiotics such as ampicillin.
Genes can be transferred between bacterial in three ways: conjugation, transduction, or transformation. Conjugation is a mating process during which genetic material is transferred from one bacterium to another of a different mating type. Transduction requires the presence of a virus to act as a carrier to transfer small pieces of DNA from one bacterium to another. Bacterial transformation involves transfer of genetic information into a cell by direct uptake of the DNA. During gene transfer, the uptake and expression of foreign DNA by a recipient bacterium can result in conferring a particular trait to a recipient lacking that trait. Transformation can occur naturally but the incidence is extremely low and is limited to relatively few bacterial strains. These bacteria can take up DNA only during the period at the end of logarithmic growth. At this time, the cells a said to be competent. Competence can be induced in E. coli with carefully controlled chemical growth conditions. Once competent, the cells are ready to accept DNA that is introduced from another source.
Plasmids can transfer genes that occur naturally within them, or plasmids can act as carriers for introducing foreign DNA from other bacteria plasmids, or even eukaryotes into recipient bacterial cells.
Materials and Procedures
I marked one sterile 15-mL tube ?+? and the other ???. I used a sterile transfer pipet to add 250 ?L of ice-cold calcium chloride to each tube and placed both tubes on the ice. I then used a sterile plastic inoculating loop to transfer a cell mass about the diameter of a pencil eraser from isolated colonies of E. coli cells from the starter plate into the + tube. I immersed these cells on the loop in the calcium chloride solution in the + tube and vigorously spun the loop in the solution to dislodge the cell mass. I then held up the tube to the light to make sure the mass had fallen off the loop and was now in the solution. I immediately suspended the cells by repeatedly pipetting in and out with a sterile transfer pipet and observed the tube under the light to make sure that there were no visible clumps of remaining cells. The suspension appeared milky white. I then returned the + tube to the ice and transferred a mass of cells into the ? tube and suspended as I had done with the + tube.
I used a sterile plastic inoculating loop to add one loopful of plasmid DNA to the + tube. When the DNA solution formed a bubble across the opening, its volume was 10 ?L. I then immersed the loopful of plasmid DNA directly into the cell suspension and spun the loop to mix the DNA with the cells. I then returned the + tube to the ice and let them incubate for 15 minutes. While the tubes were incubating, I labeled my media plates with my lab name and date. I labeled one LB/Amp plate ?+? (the experimental plate), the other was labeled LB/Amp ??? (the negative control). I then labeled the LB plates either ?+? or ???. This was a control to test the viability of the cells after they have gone through the transformation procedure.
Following the 15-minute incubation on ice, the cells were ?heat shocked?. They were removed from the ice and immediately immersed in a water bath of 42?C for 90 seconds. I gently agitated the tubes while they were in the bath and then returned them directly to the ice for 1 or more minutes. I used a sterile transfer pipet to add 250 ?L of Luria broth (LB) to each tube. I gently tapped the tubes with my finger to mix the LB with the cell suspension and placed the test tubes in a test-tube rack at room temperature for a 10-minute recovery.
I then removed some cells from each transformation tube and spread them on the plates, one plate at a time. Cells from the ? tube were spread on the ? plates, and the cells from the + tube were spread on the + plates. Using a sterile transfer pipet, I added 100 ?L of cells from the ? transformation tube to the appropriate plate(s). I immediately spread the cells over the surface of a plate using the following procedure. I slightly opened the lids (?Clam shell?) and carefully poured 4-6 beads onto each plate. By shaking it back-and-forth and up-and-down gently so that the beads moved across the entire surface, the cell suspension was evenly spread all over the agar surface. When I finished the spreading, I let the plates rest for several minutes to allow the cell suspensions to become absorbed into the agar. I then held each plate vertically over the container and slightly opened the lower part of the plates to tap out the glass beads into the container.
Using another sterile transfer pipet, I added 100 ?L of the cell suspension from the + DNA tube onto the appropriate plate(s). I immediately spread the cell suspension(s) as described in the preceding procedure. The final step was to wrap the plates together with tape and then I placed then upside down in the incubator room or at room temperature. They were incubated for approximately 24-36 hours in a 37?C incubator or 48-72 hours at room temperature.
Results
In the LB- and LB+ plates had a lawn of growth, the most growth out of all the plates. The LB/amp+ plate also showed some bacterial growth, but it was very little. The LB/amp- plate was the only plate that had no observed bacterial growth.
The total mass of plasmid (in ?L) used (used 10 ?L of plasmid at a concentration of 0.005 ?g/?L) –
Total mass = volume x concentration
= 10 ?L x .005 ?g/?L
= .05 ?g
Total volume of cell suspension prepared = CaCl2 + plasmid + Luria Broth
= 250 ?L + 10 ?L + 210 ?L
= 510 ?L
The fraction of the total cell suspension that was spread on the plate ?
Volume suspension spread / total volume suspension = fraction spread
100 ?L / 510 ?L = 0.19
The mass of plasmid in the cell suspension spread ?
Total mass plasmid x Fraction spread = mass plasmid DNA spread
.05 ?g x 0.19 = 0.0095 ?g
Discussion/Conclusion
The lawn on growth observed in the LB- and LB+ plates are due to the absence of ampicillin (antibiotic in the agar). The reason why the LB/amp+ plate showed some growth was because the cells had taken up the plasmid and had become ampicillin resistant. Since there were no plasmids to resist the ampicillin in the LB/amp- plate, there was no growth.
The purpose of plating + or ? cells on the LB plates was too see the growth and that they weren’t killed. The purpose of plating the + and ? cells on the LB/Amp plates was to observe which ones would be resistant. Since the colonies are ampicillin resistant, the transformation was successful and the plasmid is in the recipient cells. The phenotypes of the transformed colonies indicate ampicillin resistance.
If I were to conclude that the transformation was successful, I would inspect the LB/Amp+ plate because it will indicate whether the bacteria was or was not killed in heat shock and that it did pick up the plasmid.
Transformation efficiency is expressed as the number of antibiotic-resistant colonies per ?g of plasmid DNA. The object is to determine the mass of plasmid that was spread on the experimental plast and was, therefore, responsible for the transformants observed. Transformation efficiency might be affected by the picking up of enough cells, the time of cold and heat shocking, not re-suspending, and not using aseptic technique.
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