Materials List

Plating serial dilutions of E. Coli onto

TSA plates/Rifampicin plates
40 petri dish
5g NaCl
5g Tryptone
2.5g yeast extract
7.5g Agar
Deionized H2O
Rifampicin
Pipettes
Stir Rod
Erlenmeyer flask (500 ml)
Hot Plate
PBS
Test Tube Racks
Test Tubes
Light Microscope

Selecting and lysing a mutant colony

The colonies from the precious
materials list
Inoculation Loops
10 micro vials 50 l
Test Tubes
Test Tube Racks
Pipetteman 10 l
40 Pipette Tips 10 l
Thermocycling and PCR of the mutant
Bacteria Cultures from previous
step
5l mixed rpo1240F
and rpo2226R primers
20, 10 ml test tubes
Pipetteman 10 l
40 Pipette Tips 10 l
2 test tube racks
50 mL PBS Saline Solution
Forceps
1 mL Taq polymerase
Lyse-N-Go extraction reagent

Gel electrophoresis and ExoSAP

Gel electrophoresis
1% Agarose Gel
Microvials 10 l
PCR Samples
10% ExoSap
40 Pipette Tops 10 l
10 Pipetteman 10 l
PBS
Electrodes
Voltage Source
Casting Trays
(Ethidium Bromide)EtBr 0.5g/m
Microwave
Sequencing
A computer
Sequencher Data Site
Excel Sheet
A camera

Procedure & Method

Error & Conclusion

Error Analysis:
When average counts were being taken from the first hypothesis, it is evident that the
colony count do not meet expected countable amounts of 30-300 Colonies. Some of the
higher dilutions were underrepresented and this may lead to false statistics. This would
be a problem since a t test was performed to show that the first hypothesis was proven.
In the second hypothesis, error in terms of DNA Sequencing may have appeared and an
incorrect reading may have given a false diagnosis, however with the data worked with,
it was found that it was a single point mutation. Discoveries of this in the past may also
prove it to be correct that it is through a single point mutation and not through other
types.
Conclusion:
In the end of the experiment, it can be concluded that the antibiotic resistance of
Escherichia coli on the Rifampicin is caused by a single point mutation. Because of this
mutation, the transcription factors and the RNA Polymerase is able to continue on
because it the rifampicin no longer fits in the subunit groove of the bacterial DNA.
Further conclusions from this in vitro method shows that the preserved DA region
remains true, the mutation rate cannot be high around this area as the RNA Polymerase
is an essential enzyme for the bacteria. Also, reproductive stress is evident from the
results gathered from the first part in that even though the bacteria survived, the rate of
reproduction seemed to have slowed down in the environment

Abstracts, Endorsement
& Literature Cited