Purpose

The ultimate goal of this research is to look at rifampicin resistance in E. coli and to
determine how the rifampicin blocked the bacteria from performing binary fission.
Transcription factors are also looked with the bacterial RNA Polymerase and
analyzed (Fineschi, 2014). The rpo gene will be sequenced after gathering
rifampicin resistant E. coli to determine the allosteric sites of inhibition causing the
mutations within the beta subunit pocket groove (Feingold, 2011). After sequencing
the gene, the mutated DNA from the rifampicin resistant bacteria will be compared to
the strains wild type and analyzed for points of mutations using Sequencher. Further
analysis can help determine how and specifically what caused the resistance in the
E. coli.

Hypothesis & Rationale

Hypothesis 1
If E. coli is exposed to rifampicin, an antibiotic known to impede on transcription and RNA
Polymerase, then the E. coli will survive because E. coli has the ability to mutate rapidly
around the points of mutation, and as the concentration of rifampicin increase, the colonies
should decrease.
Rationale: Mutations within the rpo will cause the pocket (groove) of the bacteria to warp as
it will code for different proteins. This change of protein is enough to directly alter the
intermolecular forces that exist within the structure and the shape of the structure. With the
change of shape, rifampicin cannot bind as well in the receptor site causing the E. coli to
continue replicating.
Hypothesis 2
If the E. coli survived because of the rapid mutations, then after DNA sequencing and protein
modeling, it will be found that the allosteric site of inhibition will be changed due to one
point mutation to cause the pocket groove.
Rationale: The changes of protein cannot happen simultaneously in an allosteric groove site.
The proportion of change over the three day span is not enough to cause multiple mutations
within the rpo subunit of the protein. Also it is highly unlikely to have more than one point
of mutation because the rpo is a well preserved DNA fragment of the E. coli showing that
specific changes in this gene will allow it to undergo a microevolution.

Variables
Variables from Hypothesis 1
Independent Variable: The concentration of rifampicin used
(100 l, 200 l, 400 l, 800 l)
Dependent Variable: The colony frequency units found in each petri dish
(Measured in cfu/ml)
Control Group: The absence of rifampicin. E. coli grown without the rifampicin
solution in the TSA plates
Constants: stock concentrations used, brand of petri dish, temperature, humidity, the length
of the observations and incubation, microscope, counting methods, sterile inoculation loops,
stock solutions, incubator temperature and location, aseptic techniques, number of plates of
each group of concentrations
Variables from Hypothesis 2
Independent Variable: The DNA Sequences and protein modeling of a mutant E. coli
Dependent Variable: The type of mutation found in the rpo subunit of E. coli
Control Group: DNA Sequences and protein modeling of the wild type
(absence of mutation)
Constants: stock concentrations used, brand of petri dish, temperature, humidity, the length
of the observations and incubation, microscope, counting methods, sterile inoculation loops,
machine used, computer used, Sequencher site, angstrom view in the rotamers, PCR, agarose
sample, gel electrophoresis, ExoSAP, place of sequencing and gentotyping, incubator
location and temperature, aseptic techniques

Background Research
Rifampin specifically inhibits bacterial RNA polymerase, the enzyme responsible for
DNA transcription. So the only way for E. coli survives if exposed to Rifampicin is if it
changes its protein structure within the beta subunit of its RNA Polymerase. However it does
not do so because of Rifampicin that the E. coli mutated as mutations occur randomly. By
changing its structure, Rifampicin cannot properly bind along the receptor sites making the E.
coli strain resistant to it and continue replication and making proteins. This is because RNA
Polymerase plays a crucial role for the bacteria. It uses the DNA found in E. coli as a
template and transcribes them in the promoter region of the cell. (Gerstein, 2007).
Specifically bacterial RNA Polymerase is very important in DNA replication of the
bacteria. With DNA replication, gene expression can occur in the bacteria. In one study it was
found out by scientists that, Gene expression is linked to RNA transcription, which cannot
happen without RNA polymerase (Clancy, 2008). In all species, transcription begins with
the binding of the RNA polymerase complex (or holoenzyme) to a special DNA sequence at
the beginning of the gene known as the promoter. Activation of the RNA polymerase
complex enables transcription initiation, and this is followed by elongation of the transcript.
In turn, transcript elongation leads to clearing of the promoter, and the transcription process
can begin yet again.
With this binding into the rpo Beta subunit of E. coli. A mutation in this area will cause
the protein structure to bend or arrange itself differently making it hard for the Rifampicin to
bind as easily as it could without the mutation (Martinez, 2001). One mutation is enough to
alter the shape of the bacteria. In fact there are two types of interactions that could occur:
direct or indirect interaction. Direct interaction is if the rotamers around the protein region
bind directly to the site causing movement between the two molecules. Indirect interaction is
if the rotamers around the protein region do not necessarily bind together but however has an
influence around the region due to its electronegative state. Also the idea of Steric Hinderance
arises because if atoms are rearranged it might affect the preferred shape of the molecule and
have different amounts of rotamers based on the neighboring molecules (intermolecular
forces) and the shape of the protein (Reusch, 2013) This is important to note because one shift
can cause a chain reaction of angle bends along the molecular level warping the structure
from the original shape. This bent phenomenon can be seen by observing the rpo Beta
subunit of E. coli. (Fineschi, 2014).