1. Jason Gramling
BIOS 308
Section 1
Tim Scatterlee
June 21, 2016
Restricting E.Coli Conjugation
Abstract:
In Experiment 3, E. coli was used as our bacteria to demonstrate conjugation between
two bacteria. During conjugation one bacteria copies its own circular DNA to another bacterium.
In the experiment, Naladixic acid is used to stop conjugation at different time intervals to
determine the time of when the genes get transferred. Three genes were used, His, Pro, and Trp,
these three genes were moved via conjugation from a positive to a negative strain, thus making
the negative strain positive. Three plates were made to favor each one of the bacteria strains so
we can see when each gene enters a different strain of bacteria. Then using that time interval
data, a map of the circular DNA that was transferred can be created. The group data showed that
Trp+ gene entered around 55 minutes and His+ gene entered around 32 minutes. The Pro+ plate
yielded no colonies.
Materials and Methods: Please refer to: Johns, Mitrick A. (2010). Laboratory Manual for BIOS
308 – Genetics. DeKalb. Northern Illinois University.
8. Discussion:
In the experiment, conjugation was tested on to see how long a gene from one bacteria took to
enter a different bacterium. Conjugation is the joining of two bacteria to share circular DNA, or a
plasmid. Most often, antibiotic resistant genes are transferred from one bacteria to different
bacteria (Lin, et al., 2011). The two bacteria connect by using a structure called a pilus, which is
a tube from one bacteria called the donor cell to the other, recipient cell (Hartwell, Goldberg,
Fischer, Hood, & Aquadro, 2015). The recipient bacteria in the experiment is called a F-
bacteria, because it is missing the plasmid that was copied from F+ bacteria donors. Hfr bacteria
or high frequency of recombinants bacteria were used in this experiment as they have a high
chance of conjugation and producing recombinants (Hartwell, Goldberg, Fischer, Hood, &
Aquadro, 2015). These Hfr bacteria are used in experiments where humans are trying to
manipulate bacteria genome is some manner by inserting genes into the F+ plasmid to be copied
over to other bacteria (Ramírez-Arcos, Fernández-Herrero, Marín, & Berenguer, 1998).
Nalidixic acid was used to stop recombination at certain time intervals to give an estimate to
when each one of the three genes got transferred from the Hfr strain to the F- strain (Johns,
2010). Streptomycin was used to kill off the Hfr strains after the conjugation events started to
single out the F- bacteria (Johns, 2010).
In the experiment 50 plates were used, 2 control plates, one nutrient agar and one minimal. Then
12 plates of each type of medium used: his/trp, pro/trp, pro/his, and minimal. On the his/trp, only
Pro+ bacteria will survive; on the pro/trp, only His+ will grow; on the pro/his plate, only Trp+
will survive; and lastly the minimal plate has very few nutrients so bacteria will need the genes
to make what they need. The bacteria were first diluted in Naladixic acid for a dilution of 10-1
then a small portion was then diluted with water to produce a dilution of 10-2. Then this was
repeated every 20 minutes to 100 minutes to give us time intervals of 20 minutes. It was diluted
to lower the amount of bacteria show that the results would be recordable instead of one colony,
there would be more individual colonies (Johns, 2010). We used two different titrations to get a
better average of colonies counted.
When comparing group two data to the pooled group data, we see that the pro/his plates gave us
almost identical data results. On the group 2 data the trp+ gene entered around 55 minutes, while
on the pooled data gave an entry time of 57 minutes. However, on the plate data used, the pro/trp
plate; it was more of a difference between the two results. The group data results had a time
entry of 32 minutes while the pooled data result was about half that at 18 minutes. The pro/trp
plate had bacteria growing first at the 0 minute interval overall, and would conclude with the rest
of the data that the his+ gene was before the trp+ gene on the plasmid. Concerning pro+ gene,
the data was very inconsistent, but from the data, it looks that proline entered somewhere in the
middle of the other two.
Unfortunately the his/trp plate yielded no colonies in the group data and very little/inconsistent
counts in the pooled data, so that data was thrown out from the report. This could have been a
result of an issue or malfunction of the plates or the bacteria gene. For instance the plates we
used for the his/trp plate were replaced accidently with the minimum medium as nothing grew on
those plates either.
9. As stated above, conjugation is what most bacteria use to transfer antibiotic resistance
horizontally to other bacteria that do not have that gene, and thus making the bacteria more
deadly to humans as we try to fight bacteria that cause diseases (Lin, et al., 2011). Humans
however can also use this to their advantage in combination of other procedures, a favorable
gene can be inserted to a bacteria and that bacteria could copy said gene or trait to the other
members of the colony via conjugation. For instance humans have inserted a gene to make
insulin into bacteria, which now makes human insulin for diabetics.
From the results of the group data and the pooled data, it can be concluded that his+ gene entered
the F- bacteria sooner than the trp+ gene. This means of course that the his+ is before the trp+ gene
on the plasmid. This can be concluded from data sets where the his+ bacteria were present in the
earlier time interval plates. The equations generated from the trendlines on graphs from both of
the group data and the pooled data also supported this claim where the estimated time entry
points of the his+ gene were much earlier than the trp+ gene.
References
Hartwell, L., Goldberg, M., Fischer, J., Hood, L., & Aquadro, C. (2015). Genetics: From Genes
to Genomes. New York: McGraw-Hill.
Johns, M. A. (2010). Laboratory Manual for BIOS 308 – Genetics. DeKalb, IL, Northern Illinois
University.
Lin, A., Jimenez, J., Derr, J., Vera, P., Manapat, M. L., Esvelt, K. M., . . . Chen, I. A. (2011).
Inhibition of Bacterial Conjugation by Phage M13 and Its Protein g3p: Quantitative
Analysis and Model. PLoS One.
Ramírez-Arcos, S., Fernández-Herrero, L. A., Marín, I., & Berenguer, J. (1998). Anaerobic
Growth, a Property Horizontally Transferred by an Hfr-Like Mechanism among Extreme
Thermophiles. Journal of Bacteriology, 3137-3143.
