A.-F. Miller © 2013 Page
Cloning a Gene for Over-expression and Purification
Motivation: produce a bioremediation enzyme to metabolize herbicide residues in soil.
Nitroreductase is able to metabolize herbicides such as 2,4-dinitrophenol. However this enzyme denatures readily: is it too delicate for field work.
NADH-oxidase (NADOX) is a homologous enzyme from Thermus thermophilus that has a very high melting temperature and at 25° C displays increased catalytic activity in the presence of denaturants and high salt concentrations.
We want to over-express NADOX and test if for possible use as a bioremediation tool.
Strategy: clone the gene into an overexpression plasmid.
1 A.-F. Miller, 2013, pg
Cloning a geneA clone is a (population of) genetically identical organisms.
Plasmid, small extrachromosomal circle of DNA. (4.3 kB vs. 4.6 MB) Includes origin of replication. Often carries a set of genes needed for metabolism of occasional nutrient sources, or antibiotics.
In order for the gene to be carried over multiple generations, it needs to be replicated at least as often as the cell in which it is carried. For convenience, we clone bacterial genes into plasmids.
A.-F. Miller, 2013, pg
Simplest example
3
Cloning vectors contain an origin of replication, a selectable marker, cloning sites.Splicing a gene of interest into a plasmid provides for its propagation by host cells and permits its ready repurification (retrieval).
G&G Fig. 12.1 A.-F. Miller, 2013, pg
Cloning operations do not depend on the
content of the gene.
4
Restriction enzymesvector, insert.sticky ends / blunt endsPhosphatase, ligaseDirectional cloning behind a promotor,transformation of ‘competent’ cells, selection.
G&G Fig. 12.3, 5
A.-F. Miller, 2013, pg
Making lots of the insert: use PCR
5
PCR: Polymerase chain reaction is used to make many copies of DNA lying between the primers used.Primers are necessary in order for DNA polymerase to copy template DNA.
5’ to 3’
Template
Primer
Polymerase
A.-F. Miller, 2013, pg
PCR amplification
6
Supply template, primers (two) NTP, DNA polymerase that tolerates repeated heating (to separate DNA strands) and cooling (to permit primers to anneal onto template).
A.-F. Miller, 2013, pg 7
PCR see also Fig 12.21
+ +
+
+
+
+
+
+
+
+
++
+
+
+
+
+
+
+
+
+
+
++
+
+
linkers bind
+NTPpolymerase
melt 95°Ccool 70°C
primers at x1000
A.-F. Miller, 2013, pg
PCR, the video
8 Also http://www.youtube.com/watch?v=x5yPkxCLads
A.-F. Miller, 2013, pg 9 http://www.wehi.edu.au/education/wehitv/
DNA Replication
A.-F. Miller, 2013, pg
! Restriction enzymes are enzymes that cut DNA at specific sequences within double stranded DNA.
! Different enzymes cut DNA at different sequences.! Target sequences are usually palindromic (read the same in both
directions).! These enzymes can be used to confirm the presence of sequences by
virtue of their action of cutting DNA only when the sequence occurs.! These enzymes often cut the two DNA strands in staggered locations,
and so are also used to produce complementary overhangs “sticky ends”. ! Sticky ends allow different digestion products to be assembled together
to produce recombinant DNA molecules from two pieces that have been produced by the same restriction enzymes.We will be using Nde I (CATATG) and Hind III (AAGCTT)
5‘CGTACCGATCATATGTACCGGAT3’3‘GCATGGCTAGTATACTGGAATA5’
5‘CGTACCGATCA3‘GCATGGCTAGTAT
+
TATGTACCGGAT3’ ACATGGAATA5’
Restriction endonucleases
TATGGCTTGGAAT3’ ACCGAACCTTA5’
5‘CGTACCGATCATATGGCTTGGAAT3’3‘GCATGGCTAGTAT ACCGAACCTTA5’
Nde I
10
A.-F. Miller © 2013 Page
gene of interest
pET plasmid provides a strong promotor and other ‘designer’ features in the service of expressing a gene of interest.
Insert gene here
We choose to use a DNA polymerase that has no other duties in the cell, and that we can up-regulate at will (induce) upon addition of IPTG to the medium.(Look up IPTG)
Over-expression vectors
11 A.-F. Miller © 2013 Page
SphI (725)*
BglII (528)*T7 forwardT7
XbaI (462)*NcoI (423)*
HisTEV
NdeI (377)*EcoRI (368)
BamHI (359)*SacI (354)*
AccI (344)XhoI (328)*
T7 reverseT7 term
EcoRV (192)NheI (32)*
AccI (3624)BsmBI (3495)
ApaI (1461)*
BssHII (1661)*EcoRV (1700)
BsmBI (1865)
pBR322 origin
BsaI (4808)*
PstI (4992)*AmpR
AatII (5669)*EcoRI (5740)
pET15TEV_NESG
5741 bp
Init Sal I XhoI Nco I| Nde I EcoR I BamH I Sac I" HinD III| | TEV Protease | | | | | | | CCATGGGCCATCACCATCACCATCACgaaaacctgtattttcagagcCATATGGCGAATTCTGCGGATCCTGCGAGCTCTGTCGACGCAAAGCTTCTCGAG GGTACCCGGTAGTGGTAGTGGTAGTGcttttggacataaaagtctcgGTATACCGCTTAAGACGCCTAGGACGCTCGAGACAGCTGCGTTTCGAAGAGCTC ______6XHis tag____
Vector: pET15TEV_NESG
rbs
Our Choice:
Provides N-terminal His tag,TEV cleavage siteribosome binding site,(but no lac repressor).
12
A.-F. Miller © 2013 Page
Novagen • ORDERING 800-526-7319 • TECHNICAL SUPPORT 800-207-0144
lacI (640-1719)
ori (3153)
Ap (3
914-
4771
)
f1 origin (4903-5358)
Sty I(57)Bpu1102 I(80)
Ava I(158)Xho I(158)Eag I(166)Not I(166)Hind III(173)Sal I(179)Sac I(190)EcoR I(192)BamH I(198)
Bgl II(268)SgrA I(309)
Sph I(465)EcoN I(525)
PflM I(572)ApaB I(674)
Mlu I(990)Bcl I(1004)
BstE II(1171)Bmg I(1199)Apa I(1201)
BssH II(1401)EcoR V(1440)
Hpa I(1496)
PshA I(1835)
Psp5 II(2097)Bpu10 I(2197)
BspE I(2617)Tth111 I(2836)
Bst1107 I(2862)Sap I(2975)
BspLU11 I(3091)
AlwN I(3507)
Bsa I(4045)
Pst I(4229)
Pvu I(4354)
Sca I(4464)
Dra III(5127)
pET-21(+) (Cat. No. 69770-3) is a transcription vector designed for expression from bacterialtranslation signals carried within a cloned insert. It therefore lacks the ribosome binding site andATG start codon present on the pET translation vectors. A C-terminal His•Tag® sequence is avail-able. Unique sites are shown on the circle map. Note that the sequence is numbered by the pBR322convention, so the T7 expression region is reversed on the circular map. The cloning/expressionregion of the coding strand transcribed by T7 RNA polymerase is shown below. The f1 origin is oriented so that infection with helper phage will produce virions containing single-stranded DNAthat corresponds to the coding strand. Therefore, single-stranded sequencing should be performedusing the T7 terminator primer (Cat. No. 69337-3).
pET-21(+) cloning/expression region
TB063 12/98
pET-21(+) sequence landmarks
T7 promoter 237-253T7 transcription start 236Multiple cloning sites(BamH I - Xho I) 158-203His•Tag® coding sequence 140-157T7 terminator 26-72lacI coding sequence 640-1719pBR322 origin 3153bla coding sequence 3914-4771f1 origin 4903-5358
pET-21(+) Vector
pET-21(+)(5369bp)Eam1105 I(3984)–
T7 terminator primer #69337-3
T7 promoter
T7 promoter primer #69348-3
EcoR I Xho I
T7 terminator
BamH I Sac I Sal I Hind III Not Ilac operator
Bpu1102 I
Ava I*
Sty I
Eag IHis•Tag
Novagen • ORDERING 800-526-7319 • TECHNICAL SUPPORT 800-207-0144
lacI (640-1719)
ori (3153)
Ap (3
914-
4771
)
f1 origin (4903-5358)
Sty I(57)Bpu1102 I(80)
Ava I(158)Xho I(158)Eag I(166)Not I(166)Hind III(173)Sal I(179)Sac I(190)EcoR I(192)BamH I(198)
Bgl II(268)SgrA I(309)
Sph I(465)EcoN I(525)
PflM I(572)ApaB I(674)
Mlu I(990)Bcl I(1004)
BstE II(1171)Bmg I(1199)Apa I(1201)
BssH II(1401)EcoR V(1440)
Hpa I(1496)
PshA I(1835)
Psp5 II(2097)Bpu10 I(2197)
BspE I(2617)Tth111 I(2836)
Bst1107 I(2862)Sap I(2975)
BspLU11 I(3091)
AlwN I(3507)
Bsa I(4045)
Pst I(4229)
Pvu I(4354)
Sca I(4464)
Dra III(5127)
pET-21(+) (Cat. No. 69770-3) is a transcription vector designed for expression from bacterialtranslation signals carried within a cloned insert. It therefore lacks the ribosome binding site andATG start codon present on the pET translation vectors. A C-terminal His•Tag® sequence is avail-able. Unique sites are shown on the circle map. Note that the sequence is numbered by the pBR322convention, so the T7 expression region is reversed on the circular map. The cloning/expressionregion of the coding strand transcribed by T7 RNA polymerase is shown below. The f1 origin is oriented so that infection with helper phage will produce virions containing single-stranded DNAthat corresponds to the coding strand. Therefore, single-stranded sequencing should be performedusing the T7 terminator primer (Cat. No. 69337-3).
pET-21(+) cloning/expression region
TB063 12/98
pET-21(+) sequence landmarks
T7 promoter 237-253T7 transcription start 236Multiple cloning sites(BamH I - Xho I) 158-203His•Tag® coding sequence 140-157T7 terminator 26-72lacI coding sequence 640-1719pBR322 origin 3153bla coding sequence 3914-4771f1 origin 4903-5358
pET-21(+) Vector
pET-21(+)(5369bp)Eam1105 I(3984)–
T7 terminator primer #69337-3
T7 promoter
T7 promoter primer #69348-3
EcoR I Xho I
T7 terminator
BamH I Sac I Sal I Hind III Not Ilac operator
Bpu1102 I
Ava I*
Sty I
Eag IHis•Tag
rbs geneATG TAA
Vector: pET21
Second Choice:
Provides lac operator repress expression of toxic gene, C-terminal His tag.
13 A.-F. Miller © 2013 Page
CCATGGGCCATCACCATCACCATCACgaaaacctgtattttcagagcCATATGGCGAATTCTGCGGATCCTGCGAGCTCTGTCGACGCAAAGCTTCTCGAG
Init Sal I XhoI Nco I| Nde I EcoR I BamH I Sac I" HinD III | | TEV Protease | | | | | | |
Gene sequence TTHA0425NCBI Reference Sequence: NC_006461.1>gi|55979969:402495-403112 Thermus thermophilus HB8 chromosome, ATGGAAGCGACCCTTCCCGTTTTGGACGCGAAGACGGCGGCCCTAAAGAGGCGTTCCATCCGGCGTTACCGGAAGGACCCCGTACCCGAGGGGCTTCTCCGGGAAATCCTCGAGGCCGCCCTCCGGGCGCCCTCGGCCTGGAACCTCCAGCCCTGGCGGATCGTGGTGGTGCGGGACCCCGCCACCAAACGGGCCCTGAGGGAGGCGGCCTTCGGCCAGGCCCACGTGGAGGAGGCCCCCGTGGTCCTGGTCCTCTACGCCGACCTCGAGGACGCTCTCGCCCACCTGGACGAGGTCATCCACCCCGGGGTCCAGGGGGAAAGGCGTGAGGCGCAGAAGCAGGCCATCCAACGGGCCTTCGCCGCCATGGGGCAAGAGGCGCGAAAGGCCTGGGCCTCCGGGCAGAGCTACATCCTCTTGGGCTACCTCCTTCTCCTCCTGGAGGCTTATGGCCTCGGAAGCGTCCCCATGCTGGGGTTTGACCCCGAGAGGGTGAGGGCGATCCTGGGGCTTCCTTCCCACGCCGCCATCCCCGCCCTGGTGGCCTTGGGCTACCCGGCGGAGGAGGGCTACCCCTCCCACCGCCTGCCCCTGGAGCGGGTGGTCCTCTGGCGCTAA
NADOX gene
Cannot use Xho I
Before proposing to use Nde I, Eco RI, Hind III or Xho I,confirm that these do not cut within the gene.Search for CATATG, GAATTC, AAGCTT, CTCGAG
Eenie Meenie Minie Moe, which nuclease should we use ?
mcs of pET15TEV
Blue coloured codons are corrections I made
based on literature disagreements and the
crystal structure.14
A.-F. Miller © 2013 Page
Init Sal I XhoI Nco I| Nde I EcoR I BamH I Sac I" HinD III| | TEV Protease | | | | | | | CCATGGGCCATCACCATCACCATCACgaaaacctgtattttcagagcCATATGGCGAATTCTGCGGATCCTGCGAGCTCTGTCGACGCAAAGCTTCTCGAG GGTACCCGGTAGTGGTAGTGGTAGTGcttttggacataaaagtctcgGTATACCGCTTAAGACGCCTAGGACGCTCGAGACAGCTGCGTTTCGAAGAGCTC ______6XHis tag____
Vector: pET15TEV_NESG
Sites we will use
Therefore we will need the following flanking sequences on our primers
‘5-CATATG➝ ←TTCGAA-5’
Both of these endonucleases need some bases on either side in order to cut efficiently, so we will add a few more bases.
5’-TTTTTTCATATG➝
←TTCGAATTTTTT-5’15 A.-F. Miller © 2013 Page
Add a linker between protease cleavage and our protein.
ATGGGCCATCACCATCACCATCACgaaaacctgtattttcagagcCATATGGCGAATTCTGCGGATCCTGCGAGCTCTGTCGACGCAAAGCTTCTCG
5'3' Frame 1Met G H H H H H H E N L Y F Q S H Met A N S A D P A S S V D A K L L
http://web.expasy.org/translate/
Insert a mini-linker of flexible soluble amino acids.Gly-Ser-
Therefore add codons for Gly and Ser to gene.
GS
Intelligent choice of codons
http://muta-tion.blogspot.com/2011/07/genetic.html
What codons to use for Gly and Ser ?
Design feature for the insert (our gene)
16
A.-F. Miller © 2013 Page
Hershberg R, Petrov DA (2009) General Rules for Optimal Codon Choice. PLoS Genet 5(7): e1000556. doi:10.1371/journal.pgen.1000556
For Gly, use GGC
17 A.-F. Miller © 2013 Page
For Ser, AGC , also Welch et al (2009)
Welch M, Govindarajan S, Ness JE, Villalobos A, Gurney A, et al. (2009) Design Parameters to Control Synthetic Gene Expression in Escherichia coli. PLoS ONE 4(9): e7002. doi:10.1371/journal.pone.0007002
Arg
Leu
Ser
Ile
Note that just choosing the most common codon does not guarantee higher expression.
18
A.-F. Miller © 2013 Page
Modified early sequence of our gene.5’-TTTTTTCATATGGGCAGC➝
The authentic gene (early bases, corrections in blue, start in green)
ATGGAAGCGACCCTTCCCGTTTTGGACGCGAAGACGGCGGCCCTAAAGAGGCG
Meld together to get: (only first 30 bases to be replicated are shown)
5’-TTTTTTCATATGGGCAGCGAAGCGACCC TTCCCGTTTT GGACGCGAAG➝
How far into the gene does the primer need to extend ?Long enough that it binds selectively (short sequences can recur elsewhere in the
genome simply at random), short enough that the search for complementary sequence does not happen too slowly (fast binding and
release from wrong sites).
extension, restriction site, codons for linker
19 A.-F. Miller © 2013 Page
Hence the following “Rules of thumb” for primer design
5’ end of the gene
Restriction site permits splicing into vector. (we have the Nde I site)ATG encodes start of translation. ( √)follows a ribosome binding site and a transcription initiation site. (plasmid provides these).Extend the primer by 2-10 bases upstream of restriction site (√).Overlap with the desired gene for enough bases to give Tm ≈ 60 °C (72° preferred by some).GC content between 40-60 %.Length of 18-30 bases for specificity.NOT last three bases = G or C, not last base = T.
Tm = 2°C * (A + T) + 4°C * (C + G)http://www.basic.northwestern.edu/biotools/oligocalc.html
See http://www.embl.de/pepcore/pepcore_services/cloning/pcr_strategy/primer_design/
We will deal with these next.
20
A.-F. Miller © 2013 Page
5’-TTTTTTCATATGGGCAGCGAAGCGACCC TTCCCGTTTT GGACGCGAAG➝
Use tools on web sites to determine the GC content and melting temperatures (Tm) of duplexes predicted from different lengths of oligos.
Considering first round only when only black bases pair.
Range of Tm values account for range of Na+ concentrations that may be present (stabilizing duplex).
Considering later rounds when daughter molecules from early rounds are serving as templates.
5’-TTTTTTCATATGGGCAGCGAAGCGACCC TTCCCGTTTT GGACGCGAAG➝
49 % GC, Tm = 68 - 78 °C 39 bases
50 % GC, Tm = 67 - 74 °C 32 nucleotides But only 14 b long in round 1, 2, 64% GC, Tm 43-47 °C
50 % GC, Tm = 69 - 79 °C 40 nucleotides22 bases long in round 1, 2, 59% GC, Tm 59-67 °C.
Only 21 b in rounds 1,2, 57 % GC, Tm = 56 - 63 °C
21 A.-F. Miller © 2013 Page
5’ end of the gene
http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi
Might RNA structure mask the start site ?
√ Predicted stability of -6.2 kcal/mol (would like to have < 10).
CCATGGGCCATCACCATCACCATCACgaaaacctgtattttcagagcCATATGGGCAGCGCGAATTCTGCGGATCCTGCGAGCTCTGTCGACGCAAAGCTTCTCGAG
Init Sal I XhoI Nco I| Nde I EcoR I BamH I Sac I" HinD III | | TEV Protease | | | | | | |
Vector
5’-TTTTTTCATATGGGCAGCGAAGCGACCC TTCCCGTTTT G➝
22
A.-F. Miller © 2013 Page
Reverse primer, Rules of thumb
3’ end of the geneRestriction site permits splicing into vector.TAA stop codon (TAG and TGA are more prone to read-through).Extend the primer by 2-10 bases upstream of restriction site.Overlap with the desired gene for enough bases to give Tm ≈ 60 °C (72° preferred by some).GC content between 40-60 %.Length of 18-30 bases for specificity.NOT last three bases = G or C, not last base = T.
Check that the two primers do not pair with one another.
5’ CAC CGC CTG CCC CTG GAG CGG GTG GTC CTC TGG CGC TAG 3’
5’ CAC CGC CTG CCC CTG GAG CGG GTG GTC CTC TGG CGC TAA AAGCTT AAAAAA 3’
3’ AC GGG GAC CTC GCC CAC CAG GAG ACC GCG ATT TTCGAA TTTTTT 5’
5’ TTTTTT AAGCTT TTA GCG CCA GAG GAC CAC CCG CTC CAG GGG CA 3’
Mutate to a better stop codon. Add Hind III site and
extension.The primer is the complement strand
The primer (written right way ‘round, with site of Hind III cut)
23 A.-F. Miller © 2013 Page
Reverse primer, Rules of thumb3’ end of the geneRestriction site permits splicing into vector. (√)TAA stop codon (TAG and TGA are more prone to read-through). (√)Extend the primer by 2-10 bases upstream of restriction site. (√)Overlap with the desired gene for enough bases to give Tm ≈ 60 °C (72° preferred by some).GC content between 40-60 %.Length of 18-30 bases for specificity.NOT last three bases = G or C, not last base = T.
Check that the two primers do not pair with one another.
5’ TTTTTT AAGCTT TTA GCG CCA GAG GAC CAC CCG CTC CAG G 3’
The primer (bases that pair in the first round are in bold, all pair in rounds >2)
50 % GC Tm 67-77 °C 36 bases long (23 bases in rounds 1,2, 70 % GC and Tm 64-72 °C)45 % GC Tm 62-71 °C 31 bases long (18 bases in rounds 1,2, 67 % GC and Tm 55-61 °C)
Use green primer and a few extra cycles24
A.-F. Miller © 2013 Page
Primers for use with pET15TEV_NESG
5’-TTTTTTCATATGGGCAGCGAAGCGACCC TTCCCGTTTT G➝
5’ TTTTTT AAGCTT TTA GCG CCA GAG GAC CAC C➝ 3’
Forward
Reverse
Consequences for expression:At N-terminus, after TEV cleavage, we will add H M G S to the ‘front’ of the protein.At the C-terminus there will be no changes relative to the native protein.
Check to be sure that your two primers will not bind strongly to one-another.http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/
49 % GC, Tm = 68 - 78 °C 39 bases Only 21 b in rounds 1,2, 57 % GC, Tm = 56 - 63 °C
45 % GC Tm 62-71 °C 31 bases long (18 bases in rounds 1,2, 67 % GC and Tm 55-61 °C)
Temperatures match well.
25 A.-F. Miller © 2013 Page
Novagen • ORDERING 800-526-7319 • TECHNICAL SUPPORT 800-207-0144
lacI (640-1719)
ori (3153)
Ap (3
914-
4771
)
f1 origin (4903-5358)
Sty I(57)Bpu1102 I(80)
Ava I(158)Xho I(158)Eag I(166)Not I(166)Hind III(173)Sal I(179)Sac I(190)EcoR I(192)BamH I(198)
Bgl II(268)SgrA I(309)
Sph I(465)EcoN I(525)
PflM I(572)ApaB I(674)
Mlu I(990)Bcl I(1004)
BstE II(1171)Bmg I(1199)Apa I(1201)
BssH II(1401)EcoR V(1440)
Hpa I(1496)
PshA I(1835)
Psp5 II(2097)Bpu10 I(2197)
BspE I(2617)Tth111 I(2836)
Bst1107 I(2862)Sap I(2975)
BspLU11 I(3091)
AlwN I(3507)
Bsa I(4045)
Pst I(4229)
Pvu I(4354)
Sca I(4464)
Dra III(5127)
pET-21(+) (Cat. No. 69770-3) is a transcription vector designed for expression from bacterialtranslation signals carried within a cloned insert. It therefore lacks the ribosome binding site andATG start codon present on the pET translation vectors. A C-terminal His•Tag® sequence is avail-able. Unique sites are shown on the circle map. Note that the sequence is numbered by the pBR322convention, so the T7 expression region is reversed on the circular map. The cloning/expressionregion of the coding strand transcribed by T7 RNA polymerase is shown below. The f1 origin is oriented so that infection with helper phage will produce virions containing single-stranded DNAthat corresponds to the coding strand. Therefore, single-stranded sequencing should be performedusing the T7 terminator primer (Cat. No. 69337-3).
pET-21(+) cloning/expression region
TB063 12/98
pET-21(+) sequence landmarks
T7 promoter 237-253T7 transcription start 236Multiple cloning sites(BamH I - Xho I) 158-203His•Tag® coding sequence 140-157T7 terminator 26-72lacI coding sequence 640-1719pBR322 origin 3153bla coding sequence 3914-4771f1 origin 4903-5358
pET-21(+) Vector
pET-21(+)(5369bp)Eam1105 I(3984)–
T7 terminator primer #69337-3
T7 promoter
T7 promoter primer #69348-3
EcoR I Xho I
T7 terminator
BamH I Sac I Sal I Hind III Not Ilac operator
Bpu1102 I
Ava I*
Sty I
Eag IHis•Tag
Novagen • ORDERING 800-526-7319 • TECHNICAL SUPPORT 800-207-0144
lacI (640-1719)
ori (3153)
Ap (3
914-
4771
)
f1 origin (4903-5358)
Sty I(57)Bpu1102 I(80)
Ava I(158)Xho I(158)Eag I(166)Not I(166)Hind III(173)Sal I(179)Sac I(190)EcoR I(192)BamH I(198)
Bgl II(268)SgrA I(309)
Sph I(465)EcoN I(525)
PflM I(572)ApaB I(674)
Mlu I(990)Bcl I(1004)
BstE II(1171)Bmg I(1199)Apa I(1201)
BssH II(1401)EcoR V(1440)
Hpa I(1496)
PshA I(1835)
Psp5 II(2097)Bpu10 I(2197)
BspE I(2617)Tth111 I(2836)
Bst1107 I(2862)Sap I(2975)
BspLU11 I(3091)
AlwN I(3507)
Bsa I(4045)
Pst I(4229)
Pvu I(4354)
Sca I(4464)
Dra III(5127)
pET-21(+) (Cat. No. 69770-3) is a transcription vector designed for expression from bacterialtranslation signals carried within a cloned insert. It therefore lacks the ribosome binding site andATG start codon present on the pET translation vectors. A C-terminal His•Tag® sequence is avail-able. Unique sites are shown on the circle map. Note that the sequence is numbered by the pBR322convention, so the T7 expression region is reversed on the circular map. The cloning/expressionregion of the coding strand transcribed by T7 RNA polymerase is shown below. The f1 origin is oriented so that infection with helper phage will produce virions containing single-stranded DNAthat corresponds to the coding strand. Therefore, single-stranded sequencing should be performedusing the T7 terminator primer (Cat. No. 69337-3).
pET-21(+) cloning/expression region
TB063 12/98
pET-21(+) sequence landmarks
T7 promoter 237-253T7 transcription start 236Multiple cloning sites(BamH I - Xho I) 158-203His•Tag® coding sequence 140-157T7 terminator 26-72lacI coding sequence 640-1719pBR322 origin 3153bla coding sequence 3914-4771f1 origin 4903-5358
pET-21(+) Vector
pET-21(+)(5369bp)Eam1105 I(3984)–
T7 terminator primer #69337-3
T7 promoter
T7 promoter primer #69348-3
EcoR I Xho I
T7 terminator
BamH I Sac I Sal I Hind III Not Ilac operator
Bpu1102 I
Ava I*
Sty I
Eag IHis•Tag
rbs geneATG TAA
provide Eco RI, rbs and atg. (NdeI underlined)
GAATTCGAAGGAGATATACAT ATG GAA GCG ACC CTT CCC GTT TTG GAC G...
Forward primer
Reverse primer: as for pET15TEV
NEXT: pET21
26
A.-F. Miller © 2013 Page
provide Eco RI, rbs and atg. (NdeI underlined)
5’-TTTTGAATTCGAAGGAGATATACAT ATG GAA GCG ACC CTT CCC GTT T...
Forward primer: (rules of thumb)
Restriction site permits splicing into vector. (we have the Eco RI site)ATG encodes start of translation. ( √)Follows a ribosome binding site and a transcription initiation site. (√).Extend the primer by 2-10 bases upstream of restriction site. Overlap with the desired gene for enough bases to give Tm ≈ 60 °C (72° preferred by some).GC content between 40-60 %.Length of 18-30 bases for specificity.NOT last three bases = G or C, not last base = T.
42 bases 40% GC 66-76 °C Tm in later rounds; 17 bases 59% GC 50-55 °C Tm in rounds 1,241 bases 39% GC 65-75 °C Tm in later rounds; 16 bases 56% GC 46-51 °C Tm in rounds 1,2
Use blue primer45 bases 42% GC 67-78 °C Tm in later rounds; 20 bases 60% GC 56-63 °C Tm in rounds 1,2
27 A.-F. Miller © 2013 Page
Primers for use with pET21
5’ TTTTTT AAGCTT TTA GCG CCA GAG GAC CAC C ➝3’
Forward
Reverse
Consequences for expression:At N-terminus, The native protein will be intact (no changes).At the C-terminus there will be no changes relative to the native protein.
Check to be sure that your two primers will not bind strongly to one-another.http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/
31 bases 45 % GC Tm 62-71 °C (18 bases in rounds 1,2, 67 % GC and Tm 55-61 °C)
Temperatures match alright.
5’ TTTTGAATTCGAAGGAGATATACAT ATG GAA GCG ACC CTT CCC GT➝ 45 bases 42% GC 67-78 °C Tm in later rounds; 20 bases 60% GC 56-63 °C Tm in rounds 1,2
28
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