Mutation

in flipped nucleotides of the -10 site in dsDNA up regulates promoter

strength
Background
The purpose of this experiment is to determine the effect of a mutation on either the promoter region or the
RBS site on the gene expression. For this particular experiment, the reporter gene is the RFP within a
recreated recombinant plasmid and the fluorescence of the RFP produced under different mutations to the
promoter region shows the mutation's effect on the promoter strength. Sanger sequencing is also used to
determine the presence of the mutation and the integrity of the RFP gene. Sanger Sequencing uses a DNA
polymerase with fluorescent ddNTPs to transcribe the desired DNA at a different length, each fluorescent
dye has a different wavelength, the computer reads the nucleotides and fragments and combines them for
the sequence. The reason that the promoter is important for expression is that that the promoter regions
encode for the initiation of the transcription of the gene in the region of -10 where the sigma factor of the
DNA polymerase binds and initiates transcription (Lock and Key to Transcription: s-DNA Interaction). The
RBS is responsible for the binding of the ribosome to initiate translation after the mRNA is produced. The
consensus sequence is also present in the plasmid where it is a sequence most organisms share in common
and it is present in the -35 to -10 region after the promoter, which encodes for when to start transcription.
To measure the strength of the promoter, a reporter gene is used. A reporter gene is a gene that is attached
to the desired gene and it produces a visible change. In our experiment, we decided to perform a
substitution mutation on the -10 sites of the consensus sequence. Our hypothesis is that a substitution
mutation in the -10 regions of the gene will have a positive effect on RFP expression compared to the
original P1-RFP plasmid.

Method
For this experiment, we start with the extraction of the P1-RFP plasmids through a QIAprep plasmid
extraction protocol from experiment 2B. Then site-directed mutagenesis is performed through designed
primers for the desired mutation. For our experiment, a substitution mutation in the -10 site (TATAAT à
TAGAAT) is performed with Primer #292 as the forward primer and #333 as the reverse primer from the
SDM primer database. The primers are found through the NEB Base Changer Primer Design Program. The
primers are then introduced to the extracted P1-RFP plasmid to perform PCR. Then a KLC treatment is
performed to close the mutated plasmids and digest the original ones. The samples are then named SDM1
and SDM2 where 1 is using the original primer of each group and 2 is the primer of the neighboring group.
The plasmids are then ligated into competent cells (See ligation procedure in Lab manual). For the
competent cell, we use type DH5a and the P1 plasmids, which contains an antibiotic resistance gene for
better distinct colonies as the cells are eventually transformed onto a plate with ampicillin. The RFP used is
Biobricks part #Ba_E1010 (http://parts.igem.org/Part:BBa_E1010). After the transformation is complete,
the mutated plasmids are extracted using the QiaPrep Alkaline lysis Mini-Prep protocol from Experiment
2B and analyzed for concentration using supercoiled DNA molecules as standard as well as Nanodrop. The
sample is then diluted and sent for Sanger sequencing. The sequencing result is then used to determine
whether or not the mutation worked or not. The mutated samples are used to create three replicates and then
used for RFP analysis through a fluorometer to determine the cell density (absorbance at 660nm) and RFP
fluorescence intensity at 550 nm. This fluorescence is then normalized by dividing the cell density and
compare to the blank wells. The average fluorescence is then compared to that of P1-RFP using a t-test to
determine whether or not a change in the -10 sites will up/downregulate the promoter through gene
expression.

Results

Table 1.1 Table of Normalized RFP replicates after comparing with blank wells and divided by cell density
and means of each replicate fluorescence with the respective mutation class and expected mutation.

RFP analysis
3500000
P1-RFP
3000000
RFP fluorscence

2500000 G11 SDM2-R (p value to

P1RFP = 0.65)
2000000
G5 SDM1-R (p value to
1500000 P1RFP = 0.028)
1000000 G3 SDM1-R (p value to
P1RFP = 0.021)
500000
G7 SDM1-R (p value to
0 P1RFP = 0.89)
RFP expression

Figure 1.1 bar plot of RFP fluorescence according to the mean value with respective error bars and p-value
to the P1-RFP fluorescence, error bars are a standard deviation between the three normalized replicates

A t-test is used since comparing all group mutation data to P1-RFP.

Description of Results:
For Table 1.1 we can see that the mean of normalized RFP fluorescence for P1-RFP is 1009673 with a
standard deviation of 84491.16 and for each substitution mutation within the -10 site, the means, and
standard deviations are 298073 ± 259276.3 for TATAAT à TATCAT, 2423210 ± 455764.1 for TATAAT à
TATAAA, 2489688 ± 367782.4 for TATAAT à TAGAAT, 847829 ± 880019.5 for TATAAT à TCTAAT.
A t-test is then performed to determine the significance of each mutation compared to the P1-RFP and the
p-value for each mutation is calculated to be 0.65, 0.028, 0.021, 0.89 with a=0.05. Therefore only the
mutation for G3 and G5 proved statistical significant and are considered for comparison. And we can see
from Figure 1.1 that comparing G5 and G3 mean RFP fluorescence to the P1-RFP, we can see a significant
increase in the fluorescence, Which can indicate an increase the promoter strength. We also noticed a
significantly lower result in the G11's data with a significant error bar, which might be caused by
contamination and other factors. Table 1.2 shows the presence of the desired mutation and whether or not
the RFP sequence is still intact or not.

Conclusion:
The two only results with a statistical significance showed a significant increase in the RFP expression after
a mutation in the -10 region of the promoter and it supports our hypothesis in that a mutation in the -10
regions will up-regulate the gene expression and promoter strength. This is likely due to the function of the
-10 region of the promoter, which is the binding of the sigma factor in the polymerase to initiate the
transcription. A change in the sequence of that region will likely increase the binding specificity of the
polymerase and increase the amount of transcription, leading to an increase in RFP expression, increasing
the fluorescence (Lock and Key to Transcription: s-DNA Interaction). According to research, in order for
the RNAP to attach, the -10 sites need to be melted and the first A nucleotide and the last T nucleotide need
to be flipped to bind to the sigma factor residue. A change in the two nucleotides like in G3 and G5's
conditions will likely decrease the binding specificity of the sigma factor, leading to more nonspecific
binding and will increase the promoter strength, increasing RFP fluorescence as more transcription occurs.
As for all other point mutations in the non-flipped region, the change in nucleotide sequence might increase
the energy needed for melting and therefore leading a smaller chance of successful recognition and
transcription as melting is the necessary first step for RNAP attachment (Lock and Key to Transcription: s-
DNA Interaction). For the next experiment, I would most likely to conduct an experiment on the specific
nucleotides within the -10 region and what would each mutation in each base pair does to the up/down
regulation of the promoter strength.

Supplemental:
Mutant Name Desired Mutation Present RFP
-10 mutant Yes TATGAC Complete RFP coding sequence
found, 97% identified.
-10 mutant Yes TATCAT RFP is fully inserted with 100%
correction
-10 mutant Yes TATAAA Result: TATAAG instead of
TATAAA

-10 mutant Yes TCTAAT 99% identity between SDM1 RFP

sequence and RFP coding
sequence

Table 1.2 Describing the presence of the desired mutation, mutant position, and RFP coding sequence
integrity

Outliers (Red) are removed for comparison according to a t-test where all differences between data sets that
are statistically insignificant are ignored.