Lab 2: Natural transformation in Acinetobacter calcoaceticus

Some bacterial strains are naturally capable of taking up DNA from the environment. This is referred to as natural competence. The process requires several gene products to be present in the cell and actively expressed. Acinetobacter calcoaceticus strain BD4 and its derivatives express these genes constitutively, allowing transformation to be carried out by mixing chromosomal DNA with bacterial cells directly on a plate. If the chromosomal DNA is from Acinetobacter, then homologous recombination can occur between this DNA and the host cell genome. Recombination between wild-type and mutant DNA may convert auxotrophic mutant host cells into wild-type prototrophs. Recombination between different mutant strains may also regenerate wild-type cells, if the recombination produces transformants that have wild-type alleles for both of the mutant loci. Because the segments of DNA that are taken up during natural transformation are fairly short, this is a good technique for mapping the distance between mutations in the same operon. In this lab, you will be mapping the distance between three trp mutants by comparing the amount of prototrophic transformants produced by recombination between different mutations with the amount of transformants produced by transformation with wild-type genomic DNA. The recombination rates between genes should give you a measure of the map distance.

Acinetobacter calcoaceticus is a potential pathogen, so all work with this species must be carried out in the biohazard cabinet. All contaminated waste must be disposed of in orange disposal bags in the biohazard cabinet. Reread the aseptic technique handout before doing this lab, and follow all the steps carefully.

Laboratory Materials (per group of 3-4)

Acinetobacter calcoaceticus strain BD4 (wild-type) plate culture on LB nutrient agar

Acinetobacter calcoaceticus strain A1 (trp mutant) plate culture on LB nutrient agar

Acinetobacter calcoaceticus strain A2 (trp mutant) plate culture on LB nutrient agar

Acinetobacter calcoaceticus strain A3 (trp mutant) plate culture on LB nutrient agar

minimal salts/succinate medium plates (5 per group)

disposable plastic eppendorf tubes (4 per group)

disposable sterile plastic loops

disposable sterile cotton swabs

1 ml pipettes

p20 pipettor and tips

Sterile lysing solution

(0.050 % SDS in Standard Saline Citrate)

Laboratory Procedure – Week 1

1. A. calcoaceticus stock culture plates are provided in the Class II Biological Safety Cabinet, and will not be removed during the lab. All transfers from these plates will be carried out in the Biological Safety Cabinet, and proper aseptic technique must be followed.

2. Students will transfer a loopful of a bacterial colony from each of the 4 stock plate cultures to a different eppendorf tube, using a sterile disposable plastic loop. Try to collect similar amounts of bacteria from each stock culture colony. Loops will be disposed of in the orange plastic disposal bag in the Biological Safety Cabinet.

3. 0.5 ml of sterile lysing solution will be added to each eppendorf tube, and the tubes will be firmly capped. Tubes will be mixed to resuspend bacterial cells, and incubated at 60 ºC for 60 minutes to lyse the cells.

4. While the cell lysates are incubating, students will prepare spread plates of all 3 mutant A. calcoaceticus strains, plus two controls. Experimental plates should be labelled with the names of the strains, and controls should be labelled Control 1 and Control 2. Each plate (including the controls) should be divided up into four sections by marking the bottom with a felt pen. Each sector on the plates should be labelled with the name of one of the four A. calcoaceticus strains. Because the transfers must be done in the Biological Safety Cabinet, please label the plates ahead of time to minimise the time spent in the Safety Cabinet.

5. A small portion of a colony will be taken from each mutant stock culture plate with a sterile disposable plastic loop, and transferred to an experimental minimal media plate. The culture should then be spread over the entire surface of the plate with a sterile cotton swab. Use the same swab to spread the surface of the sector of Control 1 that is labelled with the matching strain. The fourth sector of Control 1 should be spread with wild-type A. calcoaceticus using a disposable loop and swab, in a similar manner to the experimental plates. A fresh, clean, sterile loop and swab should be used for each transfer, and disposed of in the orange plastic disposal bag. Move your plates to the side of the Biological Safety Cabinet, and leave them there until your cell lysates are ready.

6. After 60 minutes incubation, bacterial cells suspended in the lysing solution should all be killed; producing a sterile crude cell lysate containing sheared chromosomal DNA. 5 microlitres of each of these lysates will be taken from the eppendorf tubes, and evenly spread over the segment of each experimental plate and Control 2 that matches the lysate strain using a sterile swab. Use a fresh sterile swab for every individual sector of every plate to avoid cross-contamination of experimental results or cell lysate solution. Be careful not to cross the sector lines on your plates, to avoid confusing results. No cell lysate is added to Control 1, and no bacteria should be added to Control 2. Dispose of swabs in the orange plastic disposal bag.

7. Place plates in proper baskets for incubation. Plates will be incubated 24 hours at 37 °C, and then stored at 4 °C until the next lab period.

Week 2

Examine plates and count the number of colonies in each zone without opening the

plate. Report this number in a table. Express the number of prototrophic colonies

resulting from each of the transformations with mutant chromosomal DNA as a

percentage of the number of prototrophic transformants resulting from transformation

with wild-type DNA. Use this information to construct a map of the relative positions of the three mutants, using relative % transformation as a map unit. A short format report should be done for this lab.

References

Palmen, R., Hellingwerf, K.J. Uptake and processing of DNA by Acinetobacter calcoaceticus – a review. (1997) Gene 192: 179-190.