Multiple Sequence Alignment And Primer Design Of Flavodoxin Biology Essay

The purpose of this study was to plan oligonucleotide primers for a Flavodoxin protein from an unknown Desulfovibrio species. Flavodoxin protein sequences were found utilizing UniProt database and a multiple sequence alliance was performed utilizing Clustal. Oligonucleotide primers were designed to integrate two extremely conserved parts at amino acerb part 57 and 98.

Flavodoxin is of import in transporting out redox reactions within the Desulfovibrio bacterium.

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Multiple Sequence Alignment and Primer Design

Q1

To plan a set of primers capable of cloning a specific cistron, in this instance a Desulfovibrio cistron that codes for a protein called flavodoxin, it is first indispensable to choose available protein sequences from different Desulfovibrio species and so execute a multiple sequence alliance.

An appropriate database was used to turn up available Desulfovibrio flavodoxin protein sequences, in this instance UniProt KB. A hunt was performed on Desulfovibrio flavodoxin, which produces a list of flavodoxin sequences. The first entry was run in a programme located on the same database called BLASTp. The BLAST hunt found other Desulfovibrio flavodoxin protein sequences similar to the 1 of involvement. Sequences with positive alliances and low Tocopherol Values ( & lt ; 0.001 ) were selected as shown ( UniprotKB, 2010 ) .

The undermentioned FASTA sequences were downloaded from UniProt and saved as a Windows Notepad text format:

& gt ; sp|P00323| Desulfovibrio vulgaris MPKALIVYGSTTGNTEYTAETIARELADAGYEVDSRDAASVEAGGLFEGFDLVLLGCSTW

GDDSIELQDDFIPLFDSLEETGAQGRKVACFGCGDSSYEYFCGAVDAIEEKLKNLGAEIV

QDGLRIDGDPRAARDDIVGWAHDVRGAI

& gt ; sp|P71165| Desulfovibrio vulgaris ( strain Miyazaki F / DSM 19637 )

MANVLIVYGSTTGNTAWVAETVGRDIAEAGHSVEIRDAGQVEAEGLCEGRDLVLFGCSTWGDDEIELQDDFIHLYESLEATGAGKGRAACFGCGDSSYTYFCGAVDAIEERLSGLGADIV

ADSLKIDGDPRTMRDDVSAWAGRVVTAL

& gt ; sp|P80312| Desulfovibrio desulfuricans ( strive ATCC 27774 / DSM 6949 )

MSKVLILFGSSTGNTESIAQKLEELVAAGGHEVTLLNAAEASADNLADGYDAVLMGCSAW

GMEDLELQDDFAPLFDEMENMGLKGKKLAAFASGDMEYEHYCGAVPAIEEKARGLGAEVI

CEGLKIEGDASSDPDAVSAFAEDVLKKL

& gt ; sp|P18086| Desulfovibrio salexigens

MSKSLIVYGSTTGNTETAAEYVAEAFENKEIDVELKNVTDVSVADLGNGYDIVLFGCSTW

GEEEIELQDDFIPLYDSLENADLKGKKVSVFGCGDSDYTYFCGAVDAIEEKLEKMGAVVI

GDSLKIDGDPERDEIVSWGSGIADKI

& gt ; sp|Q01095| Desulfovibrio gigas

MPKALIVYGSTTGNTEGVAEAIAKTLNSEGMETTVVNVADVTAPGLAEGYDVVLLGCSTW

GDDEIELQEDFVPLYEDLDRAGLKDKKVGVFGCGDSSYTYFCGAVDVIEKKAEELGATLV

ASSLKIDGEPDSAEVLDWAREVLARV

& gt ; sp|P26492| Desulfovibrio desulfuricans

MSKVLIVFGSSTGNTESIAQKLEELIAAGGHEVTLLNAADASAENLADGYDAVLFGCSAW

GMEDLEMQDDFLSLFEEFNRIGLAGRKVAAFASGDQEYEHFCGAVPAIEERAKELGATII

AEGLKMEGDASNDPEAVASFAEDVLKQL

& gt ; sp|Q01096| Desulfovibrio gigas

MGKALVVFGSTTGNTETVAEVVAKVLEESGMAVDLKNATKVKAAGLAEGYDLVVFGCSTW

GDDEIELQEDFIPLYDDLGAAGLGGRKVAVFGCGDSSYTHFCGAVDAIAEKAASLGAKVI

DLPLKIDGAPDTAEARDWAKEVLRSAA

Q2

The seven FASTA sequences were used to execute the multiple sequence alliance. The sequences were copied into a multiple sequence alliance programme called ClustalW2.

Figure1. Results of the ClustalW2 multiple sequence alliance ( ClustalW2, 2010 ) .

Q3

The ClustalW2 alliance shows symbols that indicate parts of the aligned sequences, which are indistinguishable “ * ” , conserved permutations “ : ” and semi conserved permutations “ . ” . Figure 1 shows parts of the flavodoxin protein aligned sequence that are indistinguishable and conserved. There are besides other parts, which are non-identical and semi conserved ( ClustalW2, 2010 ) .

Three extremely conserved parts were chosen ( see figure 3 ) . Highly conserved parts consist of both indistinguishable and good conserved alliances, with really few to zero semi conserved and non indistinguishable alliances. The extremely conserved parts illustrate sequence motives of the protein, which contribute to specific biochemical maps. The sequence motives are an illustration of homology, intending that they derived from a common ascendant.

Figure3. Flavodoxin protein multiple sequence alliances and the three extremely conserved parts.

Flavodoxin is a low molecular weight ( 16kDa ) negatron transportation protein. Flavodoxin contains a non-covalently bonded flavin mononucleotide ( FMN ) prosthetic group. FMN consists of an isoalloxazine ring, which is involved in the binding of flavodoxin apoprotein and subsequent oxidation-reduction reactions ( UniprotKB, 2010 ) .

Helms et Al. identified that two aromatic residues Tryptophan and Tyrosine flank the flavin isoalloxazine ring of the flavodoxin protein. The two aromatic residues are located at aminic acids 60 and 98 severally.

Q4

As mentioned earlier, the multiple sequence alliance identified three parts that were extremely conserved. The aromatic amino acids Tryptophan 60 ‘ and Tyrosine 98 ‘ , which flank the isoalloxazine ring, are located within the conserved parts of 2 and 3 severally. The isoalloxazine ring is important to the biochemical maps of the protein and so would hold to be included in the cloning procedure. Therefore the conserved parts incorporating amino acids 60 and 98 must be incorporated into the design of the oligonucleotide primers.

Flavin isoalloxazine ring

Tryptophan 60

Tyrosine 98

Figure 4. Structure of Flavodoxin protein demoing the Flavin ring flanked by Tryptophan 60 and Tyrosine 98. Modified from RCSB Protein Data Bank, available at hypertext transfer protocol: //www.pdb.org/pdb/explore.do? structureId=3KAP.

Q5

Degenerate primers are chosen to clone the flavodoxin cistron instead than utilizing nucleotide primers, as merely the protein sequence is known in this instance.

Primers were selected from the conserved parts of 2 and 3 and included amino acids 60 and 98.

First the amino acerb sequence of the chosen part is converted into a nucleotide sequence. There are a figure of web based programmes, which can change by reversal interpret the amino acerb sequence of involvement. A programme called Molecular Toolkit was chosen.

The amino acerb sequence of the forward primer was selected from conserved part 2. Sequence “ CSTWG ” runing from amino acids 57 to 61 and so incorporates Tryptophan 60.

Amino acerb sequence: 57C S T W G61

5 ‘ TGN RWY ACY TGG GGY3 ‘

Nucleotide sequence: 5 ‘ TGN RWY ACY TGG GGY3 ‘

Forward Primer: 5’ACS RWY TGY ACC CCY3 ‘

The forward primer is complementary to the nucleotide sequence.

N = T or C

Y = T, C, A or G

R = T or A

W = C or G

S = A or G

A protein sequence can ensue in a figure of different nucleotide possibilities ( shown supra ) . The ground for this is that a big proportion of amino acids are coded by one or more codons. Serine ( s ) for illustration is coded by up to six different codons and Tryptophan ( W ) by merely one codon.

The degeneration of a primer is a theoretical figure of possible base sequences and can be calculated as follows:

1

W + M

2

F + Y + H + Q +N + K + D + E + C

3

I

4

V + P + T + A + G

6

L + S + R

The degeneration of the protein sequence CSTWG: 2A-6A-4A-1A-4 = 192

There are 192 sequence possibilities that the primer can temper to.

The complexness of the forward primer can be measured by ciphering the upper limit and minimal thaw temperatures ( Tm ) .

Equation = Tm = 2 ( A + T ) + 4 ( G + C )

Maximum Tm = 44A°I?

Minimum Tm = 52A°I?

The amino acerb sequence of the contrary primer was selected from conserved part 2. Sequence “ YTYFCG ” runing from amino acids 98 to 103 and so incorporates Tyrosine 98.

Amino acerb sequence: 98Y T Y F C G103

3’TAN ACY TAN TTN TGN GGY5 ‘

Nucleotide sequence: 3’TAN ACY TAN TTN TGN GGY5 ‘

Rearward Primer: 5’YCC SCA SAA STA SGT STA3 ‘

The contrary primer is complementary to the nucleotide sequence but in the 5 ‘ – 3 ‘ way.

N = T or C

Y = T, C, A or G

R = T or A

W = C or G

S = A or G

The degeneration of the protein sequence YTYFCG: 2A-4A-2A-2A-2A-4 = 256

There are 256 sequence possibilities that the primer can temper to.

Q6

The size of the DNA fragment amplified by the primers is calculated as follows:

Forward primer terminals at amino acid 61

Rearward primer terminals at amino acid 98

98-61 = 37 amino acids

Three bases ( triplet codon ) to one amino acid therefore: 37 A- 3 = 111 bases.

The size of the DNA fragment will be 111 bases.

The DNA fragment can be analysed by running the merchandise of the polymerase concatenation reaction ( PCR ) on an cataphoresis gel. The merchandise is added alongside an appropriate Deoxyribonucleic acid ladder, which will let the size of the merchandise to be determined after staining. Alternatively a existent – clip PCR could be undertaken, which is more sensitive.

Q7

Sequence homology is the procedure whereby cistrons and proteins have derived from a common ascendant. Sequence similarity refers to the chance of lucifer between bases or aminic acids in a sequence and may hold occurred by opportunity. Sequence similarity nevertheless does n’t intend the sequences have derived from a common ascendant. Sequence homology of two cistrons or proteins may besides portion really small similarity.

Choice parts of cistrons and proteins from sequence homology are important to the biochemical maps of the being.

Q8

Codon usage tabular arraies identify the figure of different three codons that code for specific amino acids. There are a sum of 64 different codon combinations coding for 20 aminic acids. This means that a figure of aminic acids are coded by more than one three codons e.g. Serine ( s ) is coded by up to six different codons. Codon usage tabular arraies can be obtained from databases, selected for a specific being. The codon use tabular array in this instance provides the chance of a peculiar three codon looking in the sequence of involvement. Codon usage tabular arraies were required in change overing the protein sequence into a nucleotide sequence.

Q9

It is more utile to transport out a multiple sequence alliance of a protein sequence than a nucleotide sequence for a figure of grounds. Proteins sequences are more specific compared to nucleotide sequences and do non incorporate mutants or noncoding DNAs. A little portion of a protein can be amplified to make the cryptography DNA, which may be hard to clone

via nucleotide primers. Conserved parts can besides be used to place cistron households or homologous cistrons.

Q10

Degenerate primers are designed from a protein sequence dwelling of aminic acids instead than a Deoxyribonucleic acid sequence. The primer sequence is debauched due to the places being occupied by one or more different bases.

Q11

Description: Flavodoxin

Signature: Pattern [ LIV ] – [ LIVFY ] – [ FY ] -x- [ ST ] – { V } -x- [ AGC ] -x-T- { P } -x ( 2 ) -A- { L } -x- [ LIV ] .

Entire figure of hits in UniProtKB/Swiss-Prot: 58 hits in 58 different sequences.

False positives % = 4/58 A- 100 = 6.8 %

False negatives % = 9/58 A- 100 = 15.5 %

Q12

Flavodoxin within Desulfovibrio species is involved in negatron transportation and redox reactions. In Desulfovibrio flavodoxin can replace the protein ferredoxin in S and sulfite metamorphosis ( Hrovat et al. ) .