Table of Contents

Introduction ..................................................................................................................................... 4

Aim ................................................................................................................................................. 4

Site Directed Mutagenesis | SDM ................................................................................................... 5

Plasmid Preparation | Mini-Prep ..................................................................................................... 5

Design of the Primer ....................................................................................................................... 5

Transformation ................................................................................................................................ 5

Materials ......................................................................................................................................... 6

Procedure ........................................................................................................................................ 7

Plasmid Isolation | Mini prep ...................................................................................... 7

Plasmid Quantification................................................................................................ 8

Gel Electrophoresis ..................................................................................................... 8

Primer Design ............................................................................................................. 9

Site Directed Mutagenesis ........................................................................................ 11

Transformation .......................................................................................................... 11

Results ........................................................................................................................................... 12

GFP | Chimera............................................................................................................... 12

Nanodrop....................................................................................................................... 13

Gel Results .................................................................................................................... 13

Agar Plates .................................................................................................................... 14

Discussion ..................................................................................................................................... 15

References ..................................................................................................................................... 17
Table of Figures
Figure 1: The GFP Protein. ............................................................................................................. 4
Figure 2: The GFP protein (left), The null protein (right) ............................................................ 12
Figure 3: Gel electrophoresis results............................................................................................. 13
Figure 4: Expected Results for transformation (Plasmid B) ......................................................... 14
Figure 5: GFP colonies under UV light. ....................................................................................... 14

List of Tables
Table 1: Reaction Mixture ............................................................................................................ 11
Table 2: Nanodrop absorbance measurements ............................................................................. 13
Table 3: nanodrop absorbance ratios ............................................................................................ 15
Introduction
For years scantiest have been investigating a way in which they can label protein inside
living organisms, track them, and understand the pathways and the interaction network of the
protein in target. In 2008, this was revolutionized by the discovery of new florescent protein
made by the jellyfish Aequorea Victoria. When subjected to light, jellyfish emits a green,
fluorescent light. This light emission is because of a 238 amino acid long protein called Green
Fluorescence Protein, GFP for short. The GFP protein has a barrel like structure with three
amino acids in the pocket of the barrel that govern the fluorescence activity. The three amino
acids are Serine-Tyrosine-Glycine and they are numbered 65 through 67 (Figure 1). For short
they are referred to as the Chromophore of the GFP protein. GFP is being exploited in many
medical applications as molecular markers.

Figure 1: The GFP Protein.

Aim
The aim of the report is to perform a site-directed-mutagenesis and induce a mutation in
the GFP protein. The mutation to be induced is GFP to no fluorescence represented by the
Plasmid B.

Ps: Due to an emergency, I had to travel and so I would like to mention that I started with Plasmid C,
but I had to continue my work with Plasmid B. Therefore, the report would be based on Plasmid B.
Site Directed Mutagenesis | SDM
Site Directed mutagenesis is a long protocol and is divided into mini sub-protocols. This
section will address the principle behind these protocols.

Plasmid Preparation | Mini-Prep

To start, the protein must be expressed in a bacterial system so that enough amount of the
protein is available for the results of the research to be reliable. For that purpose, plasmids are
used. Plasmids are small circular double standard DNA molecules that are independent of the
bacterial chromosome. Plasmids have marker genes such as the antibiotic resistance gene which
allow researchers to track these plasmids as bacterial cells divide. Therefore, such plasmids must
be first isolated from their original source and then quantified. [3]

Quantification of plasmids is carried out by using a high-tech quantification device called

the nanodrop that uses the principle of spectrophotometry to quantify plasmids in a given
sample. It works as follows, nucleic acids absorb UV light radiation at a specific wavelength
(260nm), and since the plasmid is made of DNA, it can be quantified by nanodrop. [4]

Design of the Primer

If we are interested in mutating a protein, then we need a starting point to change the
reading frame of the translation process. This is achieved by designing a primer that would
generate the desired mutation. The primary sequence of GFP protein can be obtained from the
protein data bank and based on the primary sequence we can generate the desired mutation. For
example, if we are interested in designing a primer that would generate the BFP mutant, then
eventually the amino acid Tyrosine(Y) at position 66 must be changed to Histidine. [5]

Transformation
After the primer design step, the plasmids must be introduced into suitable bacterial
hosts. This is usually done by transformation. The cell membrane of the host cell is chemically
modified into competent bacterial cells which can take up the Plasmid. Competent cells are
chocked by a sudden rise in temperature (heat-shock) which results in pores in the membrane so
that the Plasmid can enter the cells. [6] Positive transformations are selected on an ampicillin
containing agar plates and they are tested for fluorescence under UV light.
 In addition, this expression system selected in this experiment utilizes the Lac operon to
control gene expression. To understand how the lac operon, we need to consider two situations:

• In the absence of lactose

o When lactose (inducer) is absent, the LacI gene in the operon produces a
protein (repressor) that bind next to the promoter region and block the
transcription of RNA polymerase and therefore, protein expression.
• In the presence of lactose
o When lactose is present, it can bind the repressor protein and free the lac
operon. As a result, the RNA polymerase enzyme can be produced, and
protein expression will take place.

In this experiment, we are going to use a synthetic inducer that mimics the working
mechanism of lactose. This inducer is called IPTG and it will be provided in the agar plates to
induce protein expression. [8]

Materials
Plasmid B: GFP to no Distilled water Reaction mixture
fluorescence, Null (63T)
Agarose E. coli competent cells
Necessary buffers (BL21)
Flasks and glass wears
• P1, P2, N3, TAE Heat block and 1.5 tubes
Ethidium Bromide and
(1X), Elusion loading dye (6X) LB media, LB agar plates
buffer, PE buffer and Incubator
Microwave, Balance and
Pipettes and Tips Gel apparatus Shaker and Timer
Centrifuge GFP DNA sequence Agar Plates
Gloves and Markers PCR tubes and PrimerX online tool
Nanodrop (Thermofisher thermocycler
ExPASy tool
scientific) Ice and DPN I enzyme
Pairwise-alignment tool

Procedure

Plasmid Isolation | Mini prep

1. Previously prepared bacterial culture was incubated overnight.
2. The culture was centrifuged for 3 minutes at 8000 rpm at R.T.
3. The formed pellet was resuspended in 250 microliter P1 buffer by pipetting in a
microcentrifuge tube.
a. The P1 resuspension buffer contains the following:
i. Tris (Buffering component)
ii. RNAse A
iii. Glucose: to maintain osmotic pressure.
iv. EDTA: chelating agent to disturb the integrity of the cell membrane by
binding to calcium and magnesium ions.
4. 250 microliters of the P2 buffer were added and mixed by gentle inversions of the tube.
(4-6 times)
a. P2 is a lysis buffer, and it contains the following.
i. SDS detergent to make the membrane soluble and denature some proteins.
ii. NAOH to break the double stranded DNA by targeting Hydron bonds of
the genomic DNA.
5. 350 microliters of N3 neutralizing buffer were added and mixed by inversion.
a. N3 contains the following:
i. KOAc which neutralizes the PH.
ii. Will help in genomic DNA degradation.
6. The mixture was centrifuged for 10 mins at 13000 rpm.
7. The supernatant was transferred to a silica filter column.
8. Centrifuge the filter column again for 1 minutes and the flowthrough was discarded.
 9. 750 microliters of the PE washing buffer were added to the column and the column was
centrifuged 1 min.
10. Discard the flowthrough.
11. Centrifuge the column alone for 1 min to remove any remaining fluid.
12. Transfer the filter into a clean 1.5 ml microcentrifuge tube.
13. Add 50 microliters elution buffer to the column, wait for 1 min and centrifuge for one
min.
a. Now the plasmid is isolated, and it is supposed to be in the flowthrough.

Plasmid Quantification
1. The nanodrop software was run and the sample type was selected as DNA.
2. Apply 1.2ul Tris-EDTA buffer to the nanodrop device to generate a blank measurement.
3. Apply 1.2ul of the plasmid DNA isolated in the previous step and measure the
absorbance.

Gel Electrophoresis
Agarose Gel Preparation

1. Weigh 1 g of agarose using the balance.

2. Add 100ml of TAE (1X) buffer to make 1% agarose gel.
3. Put the flask in the microwave so that you have a homogenous clear solution.
4. Add 2 drops of Ethidium Bromide.
a. Be careful when during this step because Ethidium Bromide is carcinogenic.
5. Let the solution cool down.
6. Add the agarose into the gel apparatus and leave it until it solidifies.
7. Add TAE buffer until the gel is covered.

Gel Loading

1. Add loading dye to the DNA samples.

a. We have 6X loading dye while the gel working conditions are 1X.
b. How much of DNA and of loading dye should we add to get 1X?
 i. According to the following equation:

CiVi=CfVf

6*Vi=1X*12ul

Vi = 2ul

c. Add 2ul of 6X loading dye and 10ul of DNA.

2. Load the samples with a DNA marker into the wells of the agarose gel.
3. Run the agarose gel for 20 mins at 120V.

Primer Design

The mutagenic primer designed in this experiment should induce the deletion mutation
(63T) of threonine at position 63. Therefore, the codon for Thr at position 63 must be detected
and the corresponding DNA must be deleted. Below we have the primary sequence of the GFP
protein.

• 1 aa = 3 base pairs.
• 63 aa = 186 base pairs.
• The first bp to be deleted is the 187th.
 • Codon ACT must be deleted (yellow)

1. Go to PrimerX online tool and use this mutation code ACT187del.

2. Keep all parameters as default.
3. Click on generate primers.

This is the generated primer, but we need to double check if it actually generates the
desired mutation. For this purpose, we perform a pairwise sequence alignment between the
original GFP sequence the mutated one.

1. Go to Expasy translation tool and translate the two sequences into protein.
2. Then perform a pairwise alignment to check the difference between the two
sequences using the expasy pairwise alignment tool
3. As we see, all perfect matches except for one deletion which is our desired mutation.
Site Directed Mutagenesis
For this procedure, the reaction mix must be prepared so that in each PCR tube there is a
final volume of 50ul. Table 1.

Table 1: Reaction Mixture

Concentration
Template DNA 2ul
Forward Primer 2.5ul
Reverse Primer 2.5ul
dNTPs 1ul
10x buffer 1ul
PFU-HF DNA polymerase enzyme 5ul
dH2O 36ul

1. Add the reaction mix elements into a PCR tube from the highest to the lowest
volume.
a. Keep the polymerase for the last.
2. The site-directed mutagenesis protocol must be selected from the thermocycler, and it
should be run for two hours.
3. Once the reaction is complete, add 1 microliter of DPN enzyme and incubate at 37
degrees for an hour.
a. After the completion of the reaction, we will have both parent plasmid and
mutated one.
b. Therefore, we add the restriction enzyme DPN that recognized methylated
parent plasmid DNA and degrades it.
4. Store samples at -20 degrees.

Transformation

1. Mix 100 microliters of E. coli (BL21) competent cells with 5 microliters of plasmid
DNA.
2. Incubate on ice for 20 mins.
 3. Place them in the heat-block for 60 seconds at 42 degrees (heat-shock)
4. Place them back on ice for 2 mins
5. Add 700 microliters of LB and incubate at 37 degrees for 30 mins by shaking.
6. Pellet the cells by centrifuging for 1 min at 7000 rpm.
7. Discard the excessive LB and resuspend pellet by pipetting up and down with the fluid
that remains.
8. Apply the inoculum into agar plates containing ampicillin.
9. Spread the inoculum until it is absorbed.
10. Incubate overnight at 37 degrees.

Results
GFP | Chimera

Figure 2: The GFP protein (left), The null protein (right)

Nanodrop

Table 2: Nanodrop absorbance measurements

Nucleic acid A260 A280 260/280 260/230 Sample Factor

concentration type
(ng/µl)
23.2 0.464 0.299 1.55 0.71 DNA 50

Gel Results

Figure 3: Gel electrophoresis results

 Agar Plates

Figure 4: Expected Results for transformation (Plasmid B)

Figure 5: GFP colonies under UV light.

Discussion
As stated earlier, the purpose of this experiment is to mutate the Green Fluorescence
Protein into a Null Fluorescence protein exhibiting no fluorescence activity. This is illustrated in
figure 2 where the structure of both proteins is compared. On the left we have the native GFP
protein with its chromophore in the pocket of the barrel (green) which gives the GFP its green
glowing activity. On the right we have the structure of the GFP protein that has been mutated
into null GFP with no glowing activity. As we see in the figure, the chromophore is somehow
lost due to the deletion mutation of Thr at position 62. This absent chromophore would prevent
the glowing of the protein.

Table 2 shows the absorbance measurements of the DNA sample using the nanodrop.
DNA is absorbed at 260nm while proteins are absorbed at 280nm, the ratio between
DNA/Protein absorbance give some information about the purity of the sample (Table 2). Some
organic salts are absorbed at 230nm. As we see in table 1, the ratio 260/280 is ~1.5 which
indicates significant protein contamination in the sample. As for the 260/230 ratio it is less than
one, this means that the denominator is > than the numerator and therefore there are some
significant organic salts contamination is the sample. Unfortunately, both ratios shows that the
sample is contaminated. [7]

Table 3: nanodrop absorbance ratios

Ratio Interpretation
260/280 • ~1.8 Pure for DNA

260/230 • ~2-2.2 Pure for DNA

As for the gel results, we see that in figure 3 all groups were able to get a band in the
same position which corresponds to the plasmid. In our lane, we were able to get a decent band
which indicates that we were able to isolate the plasmid. During sample loading however, we
might have breached the gel with the tip of the pipette since some of the DNA seems to be out of
the well.
 Finally, we have the expected results for transformation in Figure 4. As reminder, the aim
was to mutate the GFP into Null GFP. So, it’s expected to see only white colonies on the plate.
In figure 4 however, we see that we have both colonies, green and white. This is probably due to
the efficiency of the DPNI restriction enzyme since it was not able to degrade all parental
plasmid harboring the GFP protein and that is why it’s expected to see colonies of both colors. In
the real results (Figure 5) however, we see that all the colonies are white and there are no green
colonies. A possible reason would be that extra amount of DPNI enzyme was added
unintentionally and maybe all parental plasmids were degraded. Another observation to notice, is
that the colonies are very small compared to the expected results, this may be some sort of a
contamination.
References

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0in,without%20the%20need%20for%20dilutions. [Accessed 12 Jun. 2022].
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Available at: https://www.fpbase.org/organism/6100/ [Accessed 12 Jun. 2022].
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[online] Available at: https://dna.uga.edu/wp-content/uploads/sites/51/2019/02/Note-on-
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8. Goldbio.com. (2016). How Does IPTG Induction Work? | GoldBio. [online] Available at:
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