My experiment objective


The objective of my experiment was to cure E. coli JM101 containing pBR plasmids that are resistant to ampicillin and tetracycline, and to observe the effect curing has on the resulting number of cultures.

I plated a culture of E. coli on a plate containing ampicillin and allowed it to grow for 48 hours as part of the curing process. I then plated 100 µL of a 10-5 and 10-6 dilution each on a control plate, a plate containing ampicillin (100µg/mL), a plate containing tetracycline (15µg/mL), and a plate containing both ampicillin (100µg/mL) and tetracycline (15µg/mL). These plates were allowed to grow for 48 hours at 37°C. The same process was repeated with a culture cured on a plate containing tetracycline. The existing cultures were counted and compared to the control plate after growth. The curing index of each plate was calculated using the formula CI = (plasmids lost/total plasmids) x 100%.

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All of the plates containing antibiotics had a larger number of cultures than the control plate, resulting in a negative curing index. This indicates that none of the plasmids were lost during the curing process in either of the antibiotics; in fact, an increase in the number of plasmids was seen after curing.

Annotation #1

Pickett MA, Everson JS, Pead PJ and Clarke IN (2005). The plasmids of Chlamydia trachomatis and Chlamydophila pneumoniae (N16): accurate determination of copy number and the paradoxical effect of plasmid-curing agents. Microbiology 151: 893 - 903

The purpose of this experiment was to establish an accurate copy number of the chlamydial plasmid in C. rachomatis and C. pneumonia, and the number of omcB genes present using quantitative PCR. Ethidium bromide, acridine orange, imipramine and novobiocin were used in attempt to remove the chlamydial plasmid from the host cells.

The bacteria were grown and incorporated into buffalo green monkey kidney cells. The infected cells were then grown on plates containing cycloheximide (1 µg/mL) and gentamicin (25 µg/mL). The buffalo green monkey kidney cells were cured using ethidium bromide, acridine orange, imipramine or novobiocin at levels below those which would be lethal to the cells. They were then incubated for 48 hours before DNA was extracted to be used in PCR analysis. Primers and probes were created for each chlamydiae species and a reaction mixture containg a forward primer (300 nM), reverse primer (300 nM), probe (100 nM) and TaqMan Universal PCR Master Mix was made. A 5 µl extraction of buffalo green monkey kidney cells was added to the reaction mixture before completion of real-time PCR in order to determine the copy number of chlamydial plasmids and the number of omcB genes.

The plasmid copy number of C. trachomatis was determined to be 4•0±0•8 plasmids per elementary body, with an initial increase and then decrease in copy number during the infectious cycle. No curing effect was seen with ethidium bromide or acridine orange alone, or imipramine and novobiocin together. This was indicated by an increase in plasmid:omcB ratio, which is equivalent to the plasmid copy number. It was determined that an increase in ethidium bromide concentration consequently increases the plasmid copy number. The plasmid copy number of C. pneumonia was determined to be 1•3±0•2 plasmids per elementary body.

In conclusion, the PCR process accurately determined the copy number of plasmids in both genera of the Chlamydiaceae. A contradictory increase in plasmid copy number was seen after the addition of all used curing agents. This was likely due to a stress response induced by action on the host cells metabolism.

The author's observation that an increase in plasmid copy number occurred after addition of curing agents will be used in my discussion. The idea of an SOS response will be used to compare to similar results that were seen in my experiment, where the number of cultures after the curing process was higher in the antibiotic media as compared to the control plate.

Annotation #2

Smith MA and Bidochka MJ (1998). Bacterial fitness and plasmid loss: the importance of culture conditions and plasmid size. Can J Microbiol 44: 351 - 355

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The intent of this experiment was to determine whether environmental conditions are able to influence the replication process of plasmids and if the size or copy number of plasmids effect its survival in various environments. The plasmid pBluescript, containing an ampicillin resistance gene, was used in this experiment.

The plasmid was transformed into E. coli XL1Blue, which also contained fungal inserts of 700 and 9000 bp. A control with no plasmid, a clone with only the plasmid pBluescript, a clone with pBluescript and a 700-bp insert, and a clone with pBluescript and a 9000-bp insert were used in this experiment. A 1:10000 dilution was made for each clone and plated on an LB agar plate containing ampicillin (75 µg/mL). The plates were incubated for 18 hours at 37°C and the colonies were counted on each plate. This was replicated five times for each clone. The control clone, 700-bp insert clone, and 9000-bp insert clone were grown in minimal media broth (with a limiting supply of glucose and salts) until they reached an optical density of 0.6 at 610 nm. They were then grown again in minimal media broth. A 1:10000 dilution was plated onto an LB plate at every third subculturing interval. These plates were again incubated for 18 hours at 37°C. The resulting colonies were then plated on an ampicillin-rich plate for the same incubation period. The colonies were counted and the percentage plasmid retention was calculated using the formula %CFU with plasmid = CFU with ampicillin/CFU without ampicillin x 100%. The CFU was compared between each of the resulting plates. The plasmid copy number was estimated using two different methods.

It was found that in minimal media broth, the lag time was longer for larger plasmids. The same was found in LB-containing plates. The quantity of colonies that contained pBluescript decreased considerably after continuous subculture. It was also found that in LB plates, the number of plasmids per bacterium decreased more drastically the larger the plasmid size. No effect was seen between the original minimal media plate and subsequent cultures in the minimal media plate. Overall, it was established that the larger the plasmid, the lower the plasmid copy number; segregational loss of plasmids occurred faster as the plasmid size increased; and subculturing in minimal media did not affect plasmid copy number. Therefore, it can be concluded that environmental conditions do affect plasmid copy number, and plasmid size does affect the ability of bacteria to survive.

The authors' observation that plasmid size and environmental conditions affect the growth of bacteria and the plasmid copy number will be cited in my introduction to establish some possible ways that plasmids can be affected, which may influence the outcome of plasmid copy number.