practical report iv- gfp mutagenesis final

Patrick Phelan 200780051

Practical Report IV- GFP Mutagenesis

Abstract

Green fluorescent protein (GFP) is a protein that emits green fluorescence at 508nm

upon excitation with light in the blue to UV range[1]. It is this unusual property that has

allowed researchers to use GFP as a reporter of expression in vivo by linking it to a

gene of interest and detecting non-invasively by fluorescence microscopy. The

protein contains a central chromophore consisting of 3 consecutive amino acids;

Serine, Tyrosine and Glycine at positions 65-67[2]. Site-directed mutagenesis of this

light-emitting group by the Quikchange method allows various mutagens, with

different light-emitting properties, to be created by transformation of X-L1

supercompetent cells with pET28c expression vectors that contain mutated GFP

constructs. For example, the Y66W mutation will give a protein that upon excitation

emits cyan fluorescence rather than green.

Introduction

GFP was first discovered and isolated in 1962 by Osamu Shimomura et.al from the

jellyfish Aequorea Victoria and was developed by Roger Tsien during the 1990’s. In

2008, Both Tsien and Shimomura were awarded the Nobel Prize in Chemistry for

their “discovery and development of green fluorescent protein.”[3] GFP has a β barrel

structure consisting of eleven β strands, with an alpha helix containing the central

chromophore (Ser65, Tyr66, Gly67) in the centre of the barrel[4], protecting it from

quenching agents such as water. The 3-residue chromophore undergoes

spontaneous oxidation and cyclisation upon reaction with molecular oxygen. Water is

eliminated and a double bond forms between the Cα & Cβ carbons of Tyr66. The

resulting structure has a network of conjugated double bonds which give the protein

its intrinsically fluorescent properties.[2] Fusion of GFP to a protein of interest can

therefore allow observation of a protein or organelle’s intracellular processes in real-

time without fixing cells and potentially damaging them. The cDNA that codes for

GFP can also be ligated to the cDNA of a gene of interest, meaning that a protein

can be expressed with the fluorescent properties of GFP whilst retaining its normal

function, making it easier to identify in vivo. Mutating the chromophore of GFP

disrupts the conjugated bond system between the amino acids, altering the

Page | 1


excitation and emission peaks of the protein. This can have a variety of benefits, for

instance if the tyrosine residue at position 66 is replaced with a tryptophan residue,

the chromophore emits light in the blue spectrum, at 485nm. This not only allows

multiple proteins to be analysed at the same time in vivo, which can allow for protein-

protein interactions to be visualised, but also gives the protein an increased stability

in a wider pH range, thus acidic organelles involved in secretory pathways e.g.

lysosomes can be studied.[5]

The aims of the experiment were;

To create a Y66W mutation in the chromophore of GFP by site-directed

mutagenesis methods such as Quikchange, changing the colour of the

fluorescence from green to cyan.

To express the CyanGFP in E.coli after transfecting a pet28C expression

vector into X1-L supercompetent cells.

To purify and detect the mutant GFP by metal affinity separation, SDS PAGE,

a Bradford assay and fluorescence spectroscopy.

Method

A comprehensive protocol can be found on pages 5-14 & 25-28 of the module

handbook.

Page | 2

Detect the presence of CyanGFP using SDS PAGE, Bradford Assay & Fluorescence spectroscopy.

Purify the protein using metal-affinity (Ni-NTA) chromatography.

Transform into E.Coli BL21(DE3)Lys to allow the mutant protein to be expressed.

Transform into X1-L supercompetent cells. Extract DNA and determine purity using NanoDrop.

Mutate GFPUV via Site-directed mutagenesis with primers designed to give the Y66W mutation.


Modifications of the protocol were as follows;

1. The minimum DNA concentration was set at 100ng/µl rather than 50ng/µl as

described in the protocol. To achieve this, an ethanol precipitation technique

was used to increase the concentration of DNA for sequencing. The reagents

required for this were 4.9µl Sodium acetate pH 5.5, 134.75µl 100% ethanol

solution and 23.05µl EB buffer.

Results

Quikchange primers were designed in order to give the Y66W mutation as desired

and they are shown in figure 1 below.

GFPuv then underwent PCR with M1 forward and reverse primers that contained the

sequences shown in figure 1. After 20-30 cycles, the PCR products were incubated

with the restriction enzyme Dpn1 which digests methylated parental DNA. The

digested DNA was then transformed into X-L1 supercompetent cells and spread on

to kanamycin plates. These were left overnight along with undigested pET28C

plasmid DNA which acted as a negative control. The following day, colonies were

counted and the results are shown in table 1 overleaf.

Page | 3

Figure 1- The Quikchange Primers designed to give the Y66W mutation in the fluorophore of GFP.


Sample Number of Colonies Transformation efficiency (cells/ng DNA)

Undigested pET28c DNA( negative control)

>200 >200

250 µl Dpn1 digest 9 2.2550 µl Dpn1 digest 54 67.5

Table 1 suggests that there was successful transformation of DNA, as both digests

had grown colonies. These were then used in a culture of E.coli for DNA extraction.

Small scale plasmid DNA was prepared for transformation, but first ethanol

precipitation, as described earlier in the method, was employed to raise the DNA

concentration from the original value of 46.1ng/µl to 100ng/µl, which was the

minimum concentration needed for efficient sequencing. The A260/A280 value,

measured by NanoDrop, was found to be 1.31, indicating that the purity of the DNA

was quite high, but a small amount of contaminants were likely present as pure DNA

should have a value between 1.8-2.

Page | 4

Table 1- Table to show the number of colonies and transformation efficiency after DPN1-digested products of the PCR were transformed into X1-L supercompetent cells

1 2 3 4 5 6 7 8

GFP bandat ~27 kDa

kDa1007058463225221711

Figure 2- SDS PAGE Photograph Showing the Presence of GFP in Lane 7. Contents are shown in Table 2 overleaf.


Well no. 1 2 3 4 5 6 7 8Fraction Cell

LysateSoluble Insoluble Unbound Wash

1Wash 2

Elution 1

Elution 2

The GFP mutant was then detected by coomassie staining and SDS PAGE. The

SDS gel photograph in figure 2 shows the presence of a band in the lane containing

elution 1 of the Ni-NTA affinity separation column. This band is roughly

correspondent to 27 kDa according to the marker, which is where GFP would be

expected to migrate to.

Discussion

The M1 forward and reverse primers were expected to cause the Y66W mutation in

the chromophore of GFP, shifting its emission peak to 485nm in the blue spectrum.

To see if the mutation had been incorporated, the small-scale plasmid DNA prepared

using the PCR products and E.coli culture was sent off to be sequenced.

Unfortunately, the sequence came back with many ‘unknown’ bases in the

sequence, rendering it unreliable. This could be due to a low DNA purity, which may

have been caused by disruption of the pellet during the preparation of the small-

scale plasmid DNA, especially as there were many spins, resulting in frequent

discarding of supernatants and resuspension of the pellet involved in the process, so

some DNA was most likely lost this way or disturbed by the pipette tip. To overcome

this problem, DNA sequencing results from another group (David Idicula and Daniel

Van) were used and this data is shown in the appendix, figures 4-7. Not only did the

sequence data show that the M1 forward and reverse primers were identical to those

described in figure 1, but also that the stop codon TAA at position 821 was deleted

and replaced with another stop codon further along the sequence (highlighted in red,

figure 6.) This confirms that a small ‘tail’ was added to the protein, without affecting

the expression of the mutant protein, which was unexpected. This was possibly

caused by DNA contaminants binding to random sites.

Both the SDS PAGE photograph (figure 2) and the fluorescence spectroscopy

readings (table 6) show that there was efficient purification and detection of GFP in

elution 1 of the Ni-NTA affinity separation. The SDS PAGE photograph shows an

isolated band at roughly 27 kDa and the fluorescence spectroscopy showed a

Page | 5

Table 2- Table to show the contents of the lanes on the SDS PAGE photograph.


reading of 100,892 for elution 1. This reading was at a wavelength of 485nm, which

is the main emission peak of the mutant GFP chromophore, indicating that the

mutation had been successful. However, the gel on the SDS PAGE photograph was

slanted and the specimens had not run very far along the gel, which could create

some unreliability in the results. This could possibly mean that the gel wasn’t run for

long enough and human error would have contributed to the slanting of the gel. Also,

elution 1 gave a very large fluorescence reading of 1,128,429 in the unbound fraction

and this was also found to have a GFP concentration of 5.76mg/ml. Because a His-

tag was fused to the GFPUV open reading frame, GFP should have bound to the Ni-

NTA column until imidazole displaced the His-tagged GFP, thus it was expected to

elute in the later fractions, not in elution 1 and not in the unbound fraction. This could

have been due to GFP aggregation and saturation of the resin, meaning GFP could

not bind and instead passed straight through the column.

Conclusion

The aims of the experiment were;

To create a Y66W mutation in the chromophore of GFP by Quikchange

mutagenesis, changing the emission peak.

To express the CyanGFP in E.coli after transfecting a pet28C expression

vector into X1-L supercompetent cells.

To purify and detect the mutant GFP by metal affinity separation, SDS PAGE,

a Bradford assay and fluorescence spectroscopy.

In regard to these aims, the experiment was relatively successful. Although the initial

DNA sequencing data was unreliable, both the Y66W mutation and the primer

sequences were found in the DNA sequencing data obtained. SDS PAGE showed

that GFP had been purified, as it was the only band in the lane and it corresponded

to roughly 27 kDa in the marker. Fluorescence spectroscopy showed that there were

very high readings for fluorescence in the first elution and unbound fractions of the

affinity separation and the fact that this was at a wavelength of 485nm shows that it

was the mutant GFP that was present. If the experiment were to be repeated,

greater care with regards to the preparation of small scale plasmid DNA would be

taken to ensure minimal disturbance of the pellet, therefore a higher purity of DNA

and a greater reliability in the sequencing data.

Page | 6


Acknowledgements

1. David Idicula and Daniel Van, DNA sequencing data. (Figures 4-7 in

appendix)

References

[1] Tsien, R. (1995) ‘Understanding, Improving and Using Green Fluorescent Proteins’, TIBS.

[2] Voet, Voet and Pratt (2013) Principles of Biochemistry. Fourth Edition.

[3] "The Nobel Prize in Chemistry 2008". Nobelprize.org. Nobel Media AB 2014. Web.

[4] Ormo, M. and Tsien, R. (1996) ‘Crystal structure of the Aequorea victoria green fluorescent protein’, Science.

[5] Sawano, A. and Miyawaki, A. (2000) ‘Directed evolution of green fluorescent protein by a new versatile PCR strategy for site-directed and semi-random mutagenesis’, Nucleic Acids Research.

Appendix and Supplementary Data

Standard (mg/ml)

2 mg/ml BSA(µl)

H2O(µl)

Elution buffer(µl)

Total Volume(µl)

0.5 100 100 200 4000.25 50 150 200 4000.1 20 180 200 4000.05 10 190 200 4000.025 5 195 200 4000.010 2 198 200 4000.005 1 199 200 4000 0 200 200 400

Page | 7

Table 3- Table to show the absorbances of the BSA standards. These were used to create Graph 1- the standard curve for the Bradford Assay


BSA Standard concentration (mg/ml)

Absorbance @ 595nm Corrected Absorbance

0.500 0.644 0.3800.250 0.509 0.2450.100 0.358 0.0940.050 0.302 0.0380.025 0.278 0.0140.010 0.257 -0.0070.005 0.276 0.0120.000 0.264 0.000

Sample Elution 1 Elution 2

Wash 1 Wash 2 Unbound Unbound Diluted

Fluorescence at 485nm

100,892 20,273 47,475 43,221 1,128,429 83,753

Unknown Fractions Absorbance at 595nm Concentration of GFP (mg/ml)

Elution 1 0.254 0.314Elution 2 0.228 0.282Wash 1 0.237 0.293Wash 2 0.234 0.289Unbound 0.918 5.76Unbound diluted 0.462 0.576

Page | 8

Graph 1- Standard Curve for the Bradford Assay of Absorbance against BSA Standard Concentration, allowing interpolation to obtain the concentrations in Table 5.

Table 6- Table to Show the Fluorescence Spectroscopy Readings at 485nm for the Various Fractions.


Page | 9

Table 5- Table to show the Absorbance and Concentration of GFP in the Fractions After Affinity Separation.

Graph 2- Graph to Show the Fluorescence and Protein Concentration for the Various Fractions.


M1F sequence: 5’-CACTTGTCACTACTTTCTCTTGGGGTGTTCAATGCTTTTCC-3’

M1R sequence: 5’-GGAAAAGCATTGAACACCCCAAGAGAAAGTAGTGACAAGTG-3’

DNA Sequence containing the forward primer binding site:

GNNNNANNCCNTNNNAATATTTTGTTNNACTTNAAGAAGGAGATATACCATGGGCAGCAGC

CATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGAGTAAAGGA

GAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCA

CAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAAT

TTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCTCTTGGGGTGTTCAATGCTTTTCCCGTTATCCGGATCATATGAAACGGCATGACTTTTTCAAGAGTGC

CATGCCCGAAGGTTATGTACAGGAACGCACTATATCTTTCAAAGATGACGGGAACTACAAGA

CGCGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATCGTATCGAGTTAAAAGGTATT

GATTTTAAAGAAGATGGAAACATTCTCGGACACAAACTCGAGTACAACTATAACTCACACAAT

GTATACATCACGGCAGACAAACAAAAGAATGGAATCAAAGCTAACTTCAAAATTCGCCACAA

CATTGAAGATGGATCCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATG

GCCCTGTCCTTTTACCAGACAACCATTACCTGTCGACACAATCTGCCCTTTCGAAAGATCCC

AACGAAAAGCGTGACCACATGGTCCTTCTTGAGTTTGTAACTGCTGCTGGGATTACACATGG

CATGGATGAGCTCTACAAAACCGCTGGCTCCGCTGCTGGTTCTGCTAGCTAATAGTGAAAG

CTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCC

CGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGG

CCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTGGCGAA

TGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGNTTACGCGCAGCGT

GACCGCTACACTTGCCAGCGCCNTAGCGCCNGNNCCTTTCGTTTNCTCCNTTCCTTNCTCG

CACGTTCGCCGGTTTTCCNGTCAAGCTCTAATCGGGGNTCCNTTTAGGNNCCGATTAAGGN

TTNACGGCCNTNNNCCCAAAAACTTGATAGGNNNNNGGTCACG

Figure 4- The M1 Forward primer caused a mutation in the DNA sequence. The primer

binding site is highlighted in grey and contains the mutations A-G and T-G.

EMBOSS_001 1 -------------------------------------------------- 0 EMBOSS_001 1 GNNNNANNCCNTNNNAATATTTTGTTNNACTTNAAGAAGGAGATATACCA 50

EMBOSS_001 1 -------------------------------------------------- 0 EMBOSS_001 51 TGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGC 100

EMBOSS_001 1 ---------ATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAAT 41 |||||||||||||||||||||||||||||||||||||||||EMBOSS_001 101 GGCAGCCATATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAAT 150

EMBOSS_001 42 TCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTG 91 ||||||||||||||||||||||||||||||||||||||||||||||||||EMBOSS_001 151 TCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTG 200

Page | 10


EMBOSS_001 92 GAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATT 141 ||||||||||||||||||||||||||||||||||||||||||||||||||EMBOSS_001 201 GAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATT 250

EMBOSS_001 142 TGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTT 191 ||||||||||||||||||||||||||||||||||||||||||||||||||EMBOSS_001 251 TGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTT 300

EMBOSS_001 192 CTCTTATGGTGTTCAATGCTTTTCCCGTTATCCGGATCATATGAAACGGC 241 |||||..|||||||||||||||||||||||||||||||||||||||||||EMBOSS_001 301 CTCTTGGGGTGTTCAATGCTTTTCCCGTTATCCGGATCATATGAAACGGC 350

EMBOSS_001 242 ATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAACGCACT 291 ||||||||||||||||||||||||||||||||||||||||||||||||||EMBOSS_001 351 ATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAACGCACT 400

EMBOSS_001 292 ATATCTTTCAAAGATGACGGGAACTACAAGACGCGTGCTGAAGTCAAGTT 341 ||||||||||||||||||||||||||||||||||||||||||||||||||EMBOSS_001 401 ATATCTTTCAAAGATGACGGGAACTACAAGACGCGTGCTGAAGTCAAGTT 450

EMBOSS_001 342 TGAAGGTGATACCCTTGTTAATCGTATCGAGTTAAAAGGTATTGATTTTA 391 ||||||||||||||||||||||||||||||||||||||||||||||||||EMBOSS_001 451 TGAAGGTGATACCCTTGTTAATCGTATCGAGTTAAAAGGTATTGATTTTA 500

EMBOSS_001 392 AAGAAGATGGAAACATTCTCGGACACAAACTCGAGTACAACTATAACTCA 441 ||||||||||||||||||||||||||||||||||||||||||||||||||EMBOSS_001 501 AAGAAGATGGAAACATTCTCGGACACAAACTCGAGTACAACTATAACTCA 550

EMBOSS_001 442 CACAATGTATACATCACGGCAGACAAACAAAAGAATGGAATCAAAGCTAA 491 ||||||||||||||||||||||||||||||||||||||||||||||||||EMBOSS_001 551 CACAATGTATACATCACGGCAGACAAACAAAAGAATGGAATCAAAGCTAA 600

EMBOSS_001 492 CTTCAAAATTCGCCACAACATTGAAGATGGATCCGTTCAACTAGCAGACC 541 ||||||||||||||||||||||||||||||||||||||||||||||||||EMBOSS_001 601 CTTCAAAATTCGCCACAACATTGAAGATGGATCCGTTCAACTAGCAGACC 650

EMBOSS_001 542 ATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGAC 591 ||||||||||||||||||||||||||||||||||||||||||||||||||EMBOSS_001 651 ATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGAC 700

EMBOSS_001 592 AACCATTACCTGTCGACACAATCTGCCCTTTCGAAAGATCCCAACGAAAA 641 ||||||||||||||||||||||||||||||||||||||||||||||||||EMBOSS_001 701 AACCATTACCTGTCGACACAATCTGCCCTTTCGAAAGATCCCAACGAAAA 750

EMBOSS_001 642 GCGTGACCACATGGTCCTTCTTGAGTTTGTAACTGCTGCTGGGATTACAC 691 ||||||||||||||||||||||||||||||||||||||||||||||||||EMBOSS_001 751 GCGTGACCACATGGTCCTTCTTGAGTTTGTAACTGCTGCTGGGATTACAC 800

EMBOSS_001 692 ATGGCATGGATGAGCTCTACAAATAA------------------------ 717 |||||||||||||||||||||| || EMBOSS_001 801 ATGGCATGGATGAGCTCTACAA--AACCGCTGGCTCCGCTGCTGGTTCTG 848

EMBOSS_001 718 -------------------------------------------------- 717 EMBOSS_001 849 CTAGCTAATAGTGAAAGCTTGCGGCCGCACTCGAGCACCACCACCACCAC 898

EMBOSS_001 718 -------------------------------------------------- 717 EMBOSS_001 899 CACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGC 948

EMBOSS_001 718 -------------------------------------------------- 717 EMBOSS_001 949 TGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGG 998

EMBOSS_001 718 -------------------------------------------------- 717 EMBOSS_001 999 TCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTGGCGAAT 1048

Page | 11


EMBOSS_001 718 -------------------------------------------------- 717 EMBOSS_001 1049 GGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGNTTACG 1098

EMBOSS_001 718 -------------------------------------------------- 717 EMBOSS_001 1099 CGCAGCGTGACCGCTACACTTGCCAGCGCCNTAGCGCCNGNNCCTTTCGT 1148

EMBOSS_001 718 -------------------------------------------------- 717 EMBOSS_001 1149 TTNCTCCNTTCCTTNCTCGCACGTTCGCCGGTTTTCCNGTCAAGCTCTAA 1198

EMBOSS_001 718 -------------------------------------------------- 717 EMBOSS_001 1199 TCGGGGNTCCNTTTAGGNNCCGATTAAGGNTTNACGGCCNTNNNCCCAAA 1248

EMBOSS_001 718 ----------------------- 717 EMBOSS_001 1249 AACTTGATAGGNNNNNGGTCACG 1271

EMBOSS_001 1 -------------------------------------------------- 0

EMBOSS_001 1 NANCTCTTNCGCTNGTAGCGCCGGATCTCGTGGTGGTGGTGGTGGTGCTC 50

EMBOSS_001 1 -------------------------------------------------- 0

Figure 5- Sequence alignment of non-mutated and mutated DNA. A-G and T-G mutation

highlighted in blue, deletion of stop codon highlighted in yellow, stop codon highlighted in

red.

DNA Sequence containing reverse primer binding site:

NANCTCTTNCGCTNGTAGCGCCGGATCTCGTGGTGGTGGTGGTGGTGCTCGAGTGCGGCC

GCAAGCTTTCACTATTAGCTAGCAGAACCAGCAGCGGAGCCAGCGGTTTTGTAGAGCTCAT

CCATGCCATGTGTAATCCCAGCAGCAGTTACAAACTCAAGAAGGACCATGTGGTCACGCTTT

TCGTTGGGATCTTTCGAAAGGGCAGATTGTGTCGACAGGTAATGGTTGTCTGGTAAAAGGA

CAGGGCCATCGCCAATTGGAGTATTTTGTTGATAATGGTCTGCTAGTTGAACGGATCCATCT

TCAATGTTGTGGCGAATTTTGAAGTTAGCTTTGATTCCATTCTTTTGTTTGTCTGCCGTGATG

TATACATTGTGTGAGTTATAGTTGTACTCGAGTTTGTGTCCGAGAATGTTTCCATCTTCTTTA

AAATCAATACCTTTTAACTCGATACGATTAACAAGGGTATCACCTTCAAACTTGACTTCAGCA

CGCGTCTTGTAGTTCCCGTCATCTTTGAAAGATATAGTGCGTTCCTGTACATAACCTTCGGG

CATGGCACTCTTGAAAAAGTCATGCCGTTTCATATGATCCGGATAACGGGAAAAGCATTGAACACCCCAAGAGAAAGTAGTGACAAGTGTTGGCCATGGAACAGGTAGTTTTCCAGTAGTG

CAAATAAATTTAAGGGTAAGTTTTCCGTATGTTGCATCACCTTCACCCTCTCCACTGACAGAA

AATTTGTGCCCATTAACATCACCATCTAATTCAACAAGAATTGGGACAACTCCAGTGAAAAGT

Page | 12


TCTTCTCCTTTACTCATATGGCTGCCGCGCGGCACCAGGCCGCTGCTGTGATGATGATGAT

GATGGCTGCTGCCCATGGTATATCTCCTTCTTAAAGTTAAACAAAATTATTTCTAGAGGGGAA

TTGTTATCCGCTCACAATTCCCCTATAGTGAGTCGTATTAATTTCGCGGGATCGAGATCTCG

ATCCTCTACGCCGGACGCATCGTGGCCGGCATCACCGGCGCCACAGGTGCGGTTGCTGGC

GCCTATATCGCCGACTTCACCGATGGGGAAGATCGGGCTCGCCACTTCGGNCTCATGAAC

GCTTGTTTCGGCGTGGGTATGGNGGCAGGCCCCTTGNCCGGGGNAATGTTGGNNGNCATC

TCCTTGGATGGACCATTCCTTGGGNNGGCGGNGNTCA

Figure 6- The DNA sequence mutated as a result of using the M1 Reverse primer. The

primer binding site is highlighted in grey and contains the T-C and A-C mutations.

EMBOSS_001 51 GAGTGCGGCCGCAAGCTTTCACTATTAGCTAGCAGAACCAGCAGCGGAGC 100

EMBOSS_001 1 ------TTATTTGTAGAGCTCATCCATGCCATGTGTAATCCCAGCAGCAG 44 | |||||||||||||||||||||||||||||||||||||||||EMBOSS_001 101 CAGCGGT--TTTGTAGAGCTCATCCATGCCATGTGTAATCCCAGCAGCAG 148

EMBOSS_001 45 TTACAAACTCAAGAAGGACCATGTGGTCACGCTTTTCGTTGGGATCTTTC 94 ||||||||||||||||||||||||||||||||||||||||||||||||||EMBOSS_001 149 TTACAAACTCAAGAAGGACCATGTGGTCACGCTTTTCGTTGGGATCTTTC 198

EMBOSS_001 95 GAAAGGGCAGATTGTGTCGACAGGTAATGGTTGTCTGGTAAAAGGACAGG 144 ||||||||||||||||||||||||||||||||||||||||||||||||||EMBOSS_001 199 GAAAGGGCAGATTGTGTCGACAGGTAATGGTTGTCTGGTAAAAGGACAGG 248

EMBOSS_001 145 GCCATCGCCAATTGGAGTATTTTGTTGATAATGGTCTGCTAGTTGAACGG 194 ||||||||||||||||||||||||||||||||||||||||||||||||||EMBOSS_001 249 GCCATCGCCAATTGGAGTATTTTGTTGATAATGGTCTGCTAGTTGAACGG 298

EMBOSS_001 195 ATCCATCTTCAATGTTGTGGCGAATTTTGAAGTTAGCTTTGATTCCATTC 244 ||||||||||||||||||||||||||||||||||||||||||||||||||EMBOSS_001 299 ATCCATCTTCAATGTTGTGGCGAATTTTGAAGTTAGCTTTGATTCCATTC 348

EMBOSS_001 245 TTTTGTTTGTCTGCCGTGATGTATACATTGTGTGAGTTATAGTTGTACTC 294 ||||||||||||||||||||||||||||||||||||||||||||||||||EMBOSS_001 349 TTTTGTTTGTCTGCCGTGATGTATACATTGTGTGAGTTATAGTTGTACTC 398

EMBOSS_001 295 GAGTTTGTGTCCGAGAATGTTTCCATCTTCTTTAAAATCAATACCTTTTA 344 ||||||||||||||||||||||||||||||||||||||||||||||||||EMBOSS_001 399 GAGTTTGTGTCCGAGAATGTTTCCATCTTCTTTAAAATCAATACCTTTTA 448

EMBOSS_001 345 ACTCGATACGATTAACAAGGGTATCACCTTCAAACTTGACTTCAGCACGC 394 ||||||||||||||||||||||||||||||||||||||||||||||||||EMBOSS_001 449 ACTCGATACGATTAACAAGGGTATCACCTTCAAACTTGACTTCAGCACGC 498

EMBOSS_001 395 GTCTTGTAGTTCCCGTCATCTTTGAAAGATATAGTGCGTTCCTGTACATA 444 ||||||||||||||||||||||||||||||||||||||||||||||||||EMBOSS_001 499 GTCTTGTAGTTCCCGTCATCTTTGAAAGATATAGTGCGTTCCTGTACATA 548

Page | 13


EMBOSS_001 445 ACCTTCGGGCATGGCACTCTTGAAAAAGTCATGCCGTTTCATATGATCCG 494 ||||||||||||||||||||||||||||||||||||||||||||||||||EMBOSS_001 549 ACCTTCGGGCATGGCACTCTTGAAAAAGTCATGCCGTTTCATATGATCCG 598

EMBOSS_001 495 GATAACGGGAAAAGCATTGAACACCATAAGAGAAAGTAGTGACAAGTGTT 544 |||||||||||||||||||||||||..|||||||||||||||||||||||EMBOSS_001 599 GATAACGGGAAAAGCATTGAACACCCCAAGAGAAAGTAGTGACAAGTGTT 648

EMBOSS_001 545 GGCCATGGAACAGGTAGTTTTCCAGTAGTGCAAATAAATTTAAGGGTAAG 594 ||||||||||||||||||||||||||||||||||||||||||||||||||EMBOSS_001 649 GGCCATGGAACAGGTAGTTTTCCAGTAGTGCAAATAAATTTAAGGGTAAG 698

EMBOSS_001 595 TTTTCCGTATGTTGCATCACCTTCACCCTCTCCACTGACAGAAAATTTGT 644 ||||||||||||||||||||||||||||||||||||||||||||||||||EMBOSS_001 699 TTTTCCGTATGTTGCATCACCTTCACCCTCTCCACTGACAGAAAATTTGT 748

EMBOSS_001 645 GCCCATTAACATCACCATCTAATTCAACAAGAATTGGGACAACTCCAGTG 694 ||||||||||||||||||||||||||||||||||||||||||||||||||EMBOSS_001 749 GCCCATTAACATCACCATCTAATTCAACAAGAATTGGGACAACTCCAGTG 798

EMBOSS_001 695 AAAAGTTCTTCTCCTTTACTCAT--------------------------- 717 ||||||||||||||||||||||| EMBOSS_001 799 AAAAGTTCTTCTCCTTTACTCATATGGCTGCCGCGCGGCACCAGGCCGCT 848

EMBOSS_001 718 -------------------------------------------------- 717

EMBOSS_001 849 GCTGTGATGATGATGATGATGGCTGCTGCCCATGGTATATCTCCTTCTTA 898

EMBOSS_001 718 -------------------------------------------------- 717

EMBOSS_001 899 AAGTTAAACAAAATTATTTCTAGAGGGGAATTGTTATCCGCTCACAATTC 948

EMBOSS_001 718 -------------------------------------------------- 717

EMBOSS_001 949 CCCTATAGTGAGTCGTATTAATTTCGCGGGATCGAGATCTCGATCCTCTA 998

EMBOSS_001 718 -------------------------------------------------- 717

EMBOSS_001 999 CGCCGGACGCATCGTGGCCGGCATCACCGGCGCCACAGGTGCGGTTGCTG 1048

EMBOSS_001 718 -------------------------------------------------- 717

EMBOSS_001 1049 GCGCCTATATCGCCGACTTCACCGATGGGGAAGATCGGGCTCGCCACTTC 1098

EMBOSS_001 718 -------------------------------------------------- 717

EMBOSS_001 1099 GGNCTCATGAACGCTTGTTTCGGCGTGGGTATGGNGGCAGGCCCCTTGNC 1148EMBOSS_001 718 -------------------------------------------------- 717 EMBOSS_001 1149 CGGGGNAATGTTGGNNGNCATCTCCTTGGATGGACCATTCCTTGGGNNGG 1198

Page | 14


EMBOSS_001 718 --------- 71

EMBOSS_001 1199 CGGNGNTCA 1207

Page | 15

Figure 7- Sequence alignment of non-mutated and mutated DNA. The Mutation is highlighted in blue and the deletion of a stop codon is highlighted in yellow.

practical report iv- gfp mutagenesis final

Documents