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---Bio 437 - Molecular Biology
Site Directed Mutagenesis of GFP
(Adapted from an Honors Project by Matthew Georgy)
Experimental
Overview
Tuesday,
Feb 6
Wednesday,
Feb 7
Tuesday,
Feb 13
Wednesday,
Feb 14
Tuesday,
Feb 21
Wed,
Feb 22
Introduction (Andy Cardillo)
Green fluorescent protein was originally isolated from the bioluminescent jellyfish Aequoria victoria. The protein is composed of 238 amino acids and requires no cofactors or substrates for fluorescence. GFP can be expressed in a variety of organisms including bacteria, yeast, Drosophilia, C. elegans, zebrafish, Xenopus, mice and humans, as well as a variety of plants. Modification of GFP has produced enhanced varieties that fluoresce thirty times greater than wild type GFP. I addition, mutation of the protein has also produced several different color variants. CLONTECH Labs has green, yellow, blue and cyan variants of GFP available, each with distinct excitation and emission spectra.
Site directed mutagenesis is a technique used to introduce specific mutations into a DNA sequence. The most common method of site directed mutagenesis involves the use of chemically synthesized oligonucleotides. Classical site directed mutagenesis involved annealing a synthetic oligonucleotide encoding the desired mutation to the template DNA, where it serves as a primer for DNA synthesis. A more efficient way to perform site directed mutagenesis involves the use of Polymerase Chain Reactions. Two mutagenic primers (forward and reverse), as well as two flanking primers (forward and reverse) are incorporated into the PCR. The first PCR cycle will produce two DNA fragments each with the desired mutation and both with a region of complementary overlap. Subsequent cycles allow these two fragments to be denatured and annealed to one another along their complementary region and serve as primers for the extension of the full length DNA sequence. This sequence is further enhanced during subsequent cycles of the PCR.
The purpose of this experiment is to utilize mutagenic primers designed specifically for the GFP gene to change the codon for amino acid 66 from TAT to CAT, which represents a change in the protein structure from Tyrosine at position 66 to Histidine. This is expected to enhance the fluorescence of the GFP protein. In addition, as second set of mutagenic primers were used to create a frameshift mutation in the GFP gene and subsequently inactivate the protein. The mutagenized PCR products were then ligated into the Bluescript plasmid and transformed into the TB1 strain of E. coli. Expression of the enhanced GFP was confirmed by fluorescence microscopy of TB1 cultures containing the recombinant plasmids. The stains containing the enhanced GFP did show increased fluorescence, while those containing the frameshift mutation showed no fluorescence. DNA sequencing of the recombinant plasmids confirmed that the changes in the DNA sequence of the GFP gene were produced as expected.Methods:
(Vicki Zubritski)
Site-Directed
Mutagenesis:
First,
colonies were incubated overnight at 37 degrees Celsius and then the bacteria
were centrifuged. The plasmid
miniprep DNA purification protocol was followed which was provided by Promega
Plus SV.
Brief
explanation of plasmid isolation and purification:
Production
of a cleared lysate:
Cells
were harvested by centrifuging and pouring off the supernatant.
Cells
were vortexed and resuspended.
Lysis
solution was added.
Alkaline
Protease solution was added to inactivate the endonucleases and other
proteins released during lysis.
Neutralization
solution was added.
Products were centrifuged.
Plasmid
Miniprep: (Wizard)
Spin
column purifed the lysate.
Centrifuged
at room temperature.
Washed
column.
Centrifuged
again at room temperature.
Repeated
wash.
Centrifuged
again.
Transfered
spin column to another tube.
Eluted
the plasmid DNA at room temperature.
Discarded
the spin column.
Stored
purified plasmid DNA at –20 degrees Celsius.
A
colony was then chosen and prepared on a wet mount.
PCR were set up with mutagenic primers by preparing a master mix and
distributing it in 5, 50 microliter reaction 0.5 milliliter tubes everything was
added except the primers.
Master
Mix:
25
microliters 10X PCR buffer
25
microliters 2.5mM dNTPs (0.25 mM final concentration)
1
microliter template DNA
125
microliters distilled water
1
microliter Taq polymerase
7.5 microliters of the appropriate primers were
then added to each tube:
Tube# Primers:
What they do
#1
– T7 + Y66HF 8/2 (date) inactivates
GFP
#2
– T3 + Y66HR 8/2
inactivates GFP
#3
- T7 + Y66HF 10/31
mutation Y66H
#4
– T3 + Y66HR 10/31
mutation Y66H
#5
– T3 + T7
amplifies the fragment
T3 à
Y66HFà
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ß
T7
35
microliters of master mix was then distributed into each of the five microfuge
tubes. Mineral oil was then added
to the tubes to prevent evaporation of products and then plasmid DNA was then
stored at –20 degrees Celsius. PCR50
thermal cycling program was then started and is as follows:
Stage
1 – 1 cycle
PCR50
Initial
denaturation
2 min. @ 94 C
Primer
anneling
45 sec. @ 50 C
Primer
extension*
1 min. @ 72 C
Standard
denaturation
45 sec. @ 94 C
Primer
anneling
45 sec. @ 50 C
Primer
extension(+)
1 min. @ 72 C
*
To improve the specificity of the DNA template concentration, anneling
temperature and Mg 2+ concentration may be varied.
(+) 1
minute extension time should be used for each kilobase pair (kbp) of product
expected.
PCR
products were purified using Quiagen Quiaquick Gel Extraction Kit Protocol.
Brief
description of purification:
·
DNA fragment was excised with scalpel.
·
Slice was weighed to determine the amount of buffer needed.
·
Gel slice was incubated at 50 Celsius to dissolve.
·
1 gel volume of isopropanol was added.
·
Spin column was added to a tube and product was centrifuged.
·
Product was washed and again centrifuged and finally, eluted.
PCR’s were set up and ran to produce a full-length mutant gene.
PCR products were then spin column purified.
The PCR products and pBS vector were then cut using XHO I (T7) and PST I
(T3) restriction enzymes. The
vector and PCR products were mixed with ligase.
Transformation screening was done by alpha-complemetation.
The Lac-Z gene à
Beta-Galactosidase (X-Gal). Products
were then plated and incubated at 37 degrees Celsius overnight.
Observations were made on the plates:
pBS à
plate with X-Gal à
colonies would be blue. PBS +
insert à
colonies would be white. Plates
were then observed and a white colony was picked and spread on a slide and
inspected using a florescence microscope. Plasmid preps were then completed to confirm the insert and
PCR was again completed to amplify the insert.
Finally, sequencing reactions were set up and ran.
1st
PCR
The initial phenotype, which was observed under an immunofluorescent
microscope, showed the pMg1 to fluoresce and the pBS did not fluoresce.
The plasmids, which were prepared from the pBS and pMg1, worked well and
had good yields as seen below in Figure 1.
Figure 1.
Bste
pBS pMg1
pBS
pMg1
Bste
Marker
Marker
As can be seen in the above figure, the pBS migrated faster then the pMg1. In comparison with the Bste marker, it appears that pBS has approximately 4300 bp and pMg1 has approximately 4800 bp. These results were expected.
PCR was done on these samples, pBS and pMg1, but was unsuccessful due to nonspecific and unexpected bands as well as different size fragments as can be seen in Figure 2.
Figure 2.
Bste 8/2
8/2 10/31
10/31 Control
Marker T7 T3
T7 T3
T3+T7
Due to the results from the first PCR a second attempt was required to proceed with the experiment. Figure 3 is the second attempt at PCR yielded good results and was used for cloning. The lane order was as follows: Lane 1 was the 8/2 T3+T7 control, Lane 2 was 8/2 T7, Lane 3 was 10/31 T7, Lane 4 was the 10/31 T3+T7 control, Lane 5 was the Bste Marker, Lane 6 was 8/2 T3, and lane 7 was 10/31 T3. The results for the size of the samples in each lane were: controls at approximately 800 bp, the T7 at approximately 600 bp, and the T3 at approximately 400 bp. See figure 3 on next page. Once the 8/2 and 10/31 were cloned, they were wet mounted and observed under an immunofluorescent microscope. This PCR made 2 fragments, 1 of T3 and 1 of T7, that overlapped at the mutation in Y66HF and Y66HR in the forward and reverse primers.
Figure 3
Control
8/2
10/31
Control
Bste
8/2
10/31
T3+T7
T7
T7
T3+T7
Marker
T3
T3
8/2
10/31
A second PCR was then done to make a full-length product in which The T7 fragment and the T3 fragment were joined. This can be seen in Figure 4.
Figure 4.
These were then used to clone
into pBS. The clones were wet
mounted and observed under immunofluorescent microscopy.
The 10/31 was observed fluorescing and the 8/2 did not fluoresce.
The cloned samples were then isolated and used for a PCR that was
Figure
5. Lane 1 8/2 and
Lane 2 10/31
used for DNA sequencing which can be seen in Figure 5.
Mutations were observed in the 8/2 T3, 8/2 Reverse, and 10/31 Forward. The sequence that was suppose to be found was TACTTTCTCTTATGG. The mutation that was found for 8/2 T3 was TACTTTACTCATATGG. This can be seen in Figure 6.
Figure 6.
The mutation found in 8/2 Reverse was TACTTTCACTCATGG. This can be seen in Figure 7.
Figure 7.
Figure
8.
The mutation found with 10/31 Reverse was TACTTTCACTCATGG.
This can be seen in Figure 8.
Wendilorion Meyers Discussion (SDM)
This experiment resulted in mutations in the sequences of the 8/2 T3 clone, the 8/2 Reverse clone, and the 10/31 Forward clone. After analysis of sequence gels, it was found that the 8/2 T3 clone mutated from TACTTTCTCTTATGG to TACTTTACTCATATGG. The 8/2 Reverse clone mutated to TACTTTCACTCATGG. The 10/31 Reverse clone mutated to TACTTTCACTCATGG.
The 10/31 clone mutations occurred where we anticipated. We were unsure as to where the mutations would occur in the 8/2 clones, as there were a variety of mutations due to our preparation of the sample. The end result of these mutations was the fluorescing of the 10/31 clones and the non-fluorescent nature of the 8/2 clones. Both these results were expected due to the mutation site in the 10/31 sample and the multiple mutations in the 8/2 clones, which yielded the gene non-functional.