Ghosh LabUniversity of Arizona

Department of Chemistry

Outline

•Cloning overview

•pDRAW32

• Design

•Gene

•Insert

•Primers

•Further considerations (optimization of the process)

•Transformation

Cloning OverviewFour main steps in cloning:

•Insert synthesis

•Restriction enzyme digest

•Ligation

•Transformation

+Functionalconstruct

Plasmid(vector)

Insert(your gene)

Design OverviewSteps to follow in designing your cloning experiment:

•Design your gene

•Design your insert

•Pick your enzymes

•Check your design

•Recheck your design

Functionalconstruct

All of the important information in one place!

pDRAW32Plasmid maps: pDRAW32

pETDuet GFPuv6104 bp

XbaI - 30 - T'CTAG_ANcoI - 69 - C'CATG_G

BamHI - 106 - G'GATC_CEcoRI - 112 - G'AATT_C

NcoI - 281 - C'CATG_GMscI - 286 - TGG'CCA

NdeI - 345 - CA'TA_TGBsrGI - 391 - T'GTAC_AMluI - 437 - A'CGCG_TBaeI - 456 - ACnnnnGTAyCnnnnnnn_nnnnn'BaeI - 489 - GrTACnnnnGTnnnnnnnnnn_nnnnn'PspXI - 536 - vC'TCGA_GbAvaI - 536 - C'yCGr_GXhoI - 536 - C'TCGA_GBstZ17I - 565 - GTA'TACBamHI - 635 - G'GATC_CMfeI - 675 - C'AATT_GSalI - 718 - G'TCGA_CHincII - 720 - GTy'rACBstBI - 737 - TT'CG_AAEcoICRI - 820 - GAG'CTCSacI - 822 - G_AGCT'CNotI - 834 - GC'GGCC_GCAflII - 847 - C'TTAA_GBsrGI - 874 - T'GTAC_ANdeI - 982 - CA'TA_TGBglII - 989 - A'GATC_TMfeI - 995 - C'AATT_GEcoRV - 1003 - GAT'ATCFseI - 1012 - GG_CCGG'CCBaeI - 1016 - GrTACnnnnGTnnnnnnnnnn_nnnnn'AsiSI - 1021 - GCG_AT'CGCPvuI - 1021 - CG_AT'CGZraI - 1028 - GAC'GTCAatII - 1030 - G_ACGT'CAcc65I - 1032 - G'GTAC_CKpnI - 1036 - G_GTAC'CPspXI - 1038 - vC'TCGA_GbAvaI - 1038 - C'yCGr_GXhoI - 1038 - C'TCGA_GBaeI - 1049 - ACnnnnGTAyCnnnnnnn_nnnnn'PflMI - 1085 - CCAn_nnn'nTGGPacI - 1113 - TTA_AT'TAAAvrII - 1117 - C'CTAG_GBlpI - 1135 - GC'TnA_GCBsaAI - 1460 - yAC'GTrDraIII - 1463 - CAC_nnn'GTGAloI - 1499 - GAACnnnnnnTCCnnnnnnn_nnnnn'BsaXI - 1499 - ACnnnnnCTCCnnnnnnn_nnn'BsaXI - 1529 - GGAGnnnnnGTnnnnnnnnn_nnn'AloI - 1531 - GGAnnnnnnGTTCnnnnnnn_nnnnn'PsiI - 1588 - TTA'TAASspI - 1668 - AAT'ATT

AhdI - 1873 - GACnn_n'nnGTCBsaI - 1934 - GGTCTCn'nnnn_

BglI - 1993 - GCCn_nnn'nGGCFspI - 2095 - TGC'GCA

PvuI - 2243 - CG_AT'CGScaI - 2353 - AGT'ACT

XmnI - 2472 - GAAnn'nnTTCBssSI - 2537 - C'ACGA_G

AlwNI - 3069 - CAG_nnn'CTGBssSI - 3305 - C'ACGA_G

PciI - 3478 - A'CATG_T

SapI - 3595 - GCTCTTCn'nnn_

BstZ17I - 3711 - GTA'TACBsaAI - 3730 - yAC'GTr

Tth111I - 3736 - GACn'n_nGTCPfoI - 3835 - T'CCnGG_A

BsmBI - 3837 - CGTCTCn'nnnn_

XmnI - 3924 - GAAnn'nnTTC

Afe I - 4228 - AGC'GCT

AlfI - 4292 - GCAnnnnnnTGCnnnnnnnnnn_nn'

Bpu10I - 4373 - CC'TnA_GC

PpuMI - 4473 - rG'GwC_Cy

AlfI - 4513 - GCAnnnnnnTGCnnnnnnnnnn_nn'

BsaXI - 4674 - ACnnnnnCTCCnnnnnnn_nnn'BsaXI - 4704 - GGAGnnnnnGTnnnnnnnnn_nnn'

BsmBI - 4727 - CGTCTCn'nnnn_

HincII - 4840 - GTy'rACHpaI - 4840 - GTT'AAC

BssHII - 4931 - G'CGCG_C

PspOMI - 5135 - G'GGCC_CApaI - 5139 - G_GGCC'CBstEII - 5160 - G'GTnAC_C

BclI - 5328 - T'GATC_A - dam methylated!MluI - 5342 - A'CGCG_T

BstAPI - 5666 - GCAn_nnn'nTGCPflMI - 5767 - CCAn_nnn'nTGG

PfoI - 5774 - T'CCnGG_AEcoNI - 5810 - CCTnn'n_nnAGG

SphI - 5875 - G_CATG'CAfeI - 5941 - AGC'GCTSgrAI - 6023 - Cr'CCGG_yG

ClaI - 6067 - AT'CG_AT - dam methylated!

GFPuv

AMP

lacI

pDRAW32

You can look at the sequence in detail

•Open reading frames

•Translation

•Restriction sites

•Complementary strand

Design of the Gene

Example, the gene we want:G C D R A S P Y C G

We got this from phage display:ggctgcgacagggcgagcccgtactgcggtG C D R A S P Y C G

Phage sequence

Final sequence for the gene of interest:ggctgcgacagggcgagcccgtactgcggttaaG C D R A S P Y C G *

Add a stop codon

If you are cloning out of a known plasmid, just use the sequence that you have

Design of the Gene

•If you are designing the gene from scratch, keep in mind codon usage

•Not all codons are created equal

•Un-optimized codons could lead to lower expression levels

•The codon usage reflects levels of tRNA available in E. Coli

•Pay attention to the stop codons too (XL1-Blues read through TAG {amber stop codon} 20% of the time)

http://www.bioinformatics.org/sms2/rev_trans.html

http://www.entelechon.com/index.php?id=tools/backtranslation&lang=eng

or preferably…

What if we don’t have the DNA sequence?Design from scratch! (don’t forget about codon usage)E. Coli Codon Usage

UUU F 0.59 UCU S 0.17 UAU Y 0.6 UGU C 0.47UUC F 0.41 UCC S 0.15 UAC Y 0.4 UGC C 0.53UUA L 0.15 UCA S 0.15 UAA * 0.6 UGA * 0.31UUG L 0.13 UCG S 0.13 UAG * 0.09 UGG W 1

CUU L 0.12 CCU P 0.19 CAU H 0.58 CGU R 0.35CUC L 0.1 CCC P 0.13 CAC H 0.42 CGC R 0.34CUA L 0.04 CCA P 0.21 CAA Q 0.34 CGA R 0.07CUG L 0.46 CCG P 0.47 CAG Q 0.66 CGG R 0.12

AUU I 0.49 ACU T 0.19 AAU N 0.51 AGU S 0.16AUC I 0.38 ACC T 0.38 AAC N 0.49 AGC S 0.23AUA I 0.13 ACA T 0.19 AAA K 0.73 AGA R 0.08AUG M 1 ACG T 0.24 AAG K 0.27 AGG R 0.05

GUU V 0.29 GCU A 0.19 GAU D 0.63 GGU G 0.34GUC V 0.2 GCC A 0.26 GAC D 0.37 GGC G 0.36GUA V 0.17 GCA A 0.24 GAA E 0.67 GGA G 0.14GUG V 0.34 GCG A 0.31 GAG E 0.33 GGG G 0.16

•Endonucleases (or restriction enzymes) are enzymes which cut DNA at specific internal recognition sequences

•Compare to exonucleases, which cut from one end

•You must choose restriction sites that are available in the plasmid you are cloning into

•They must not appear in your gene (silent mutation can remove unwanted sites in your designed gene)

Choice of Restriction Sites/Enzymes

Once you have your gene, you need to design a way to get it into your plasmid

•Restriction sites must exist only once in your plasmid

•They must be in the correct position relative to the purification tag

•Restrictions sites usually add extra residues to your gene product; make sure they are compatible with your peptide/protein

•Some restriction sites are sub-optimal for cloning

•Blunt end sites

•dam and dcm methylation-affected enzymes

Really Important Factors to Remember When Choosing Restriction Enzymes

AGCCAG GATCCGGGCTGCAAGCGGTTAAG AATTCGTCGACGTCGACG AATTCTTAACCGCTTCCAGCCCG GATCCTGGCT

GATCCGGGCTGCAAGCGGTTAAG AATTCTTAACCGCTTCCAGCCCG

GATCCTGGCTAGCCAG AATTCGTCGACGTCGACG+

“sticky ends”

AGCCAGAT ATCGGGCTGCAAGCGGTTAACAG CTGGTCGACGTCGACCAG CTGTTAACCGCTTCCAGCCCGAT ATCTGGCT

ATCGGGCTGCAAGCGGTTAACAGCTGTTAACCGCTTCCAGCCCGAT

AGCCAGATATCTGGCT + CTGGTCGACGTCGACCAG

•“Sticky ends”: 5’ or 3’ over-hangs that allow the DNA to anneal even though it is not covalently bound

•Help with the next step: ligation

Most common restriction enzymes

Blunt-end restriction enzymes No sticky ends

Blunt vs Sticky Ends

Digestion

Digestion

dam Methylation

NO

N

N

ON

NHO

P

P

O

O O

O

O

O

MeNO

N

N

ON

NH2

O

P

P

O

O O

O

O

O

Dam methylase

•Dam methylase puts a methyl group on the nitrogen of 6th position of adenosine at the site: GATC

•All of the E. Coli that we use generate DNA with dam methylation

•Some enzymes only cut dam methylated DNA: eg DpnI

•Some enzymes do not cut dam methylated DNA: eg XbaI

http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/dam_dcm_methylases_of_ecoli.asp

dcm Methylation

http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/dam_dcm_methylases_of_ecoli.asp

Dcm methylase

O

P

O

O O

O NN

NH2

O

O

P

O

O O Me

O

P

O

O O

O NN

NH2

O

O

P

O

O O

•Dcm methylase puts a methyl group on the carbon of 5th position of cytidine at the site: CCAGG and CCTGG

•The enzyme we use most that can be affected by dcm methylation is SfiI

•XL1-Blues and BL21s are both Dcm+

•Once you have your restriction enzymes chosen, it is time to design the final complete gene

•The multiple cloning site (or whatever plasmid you are cloning into) should already have the 5’ portion of the gene intact (i.e. RBS, spacer, Met)

• Sequences must be in frame

NcoI BtgI51 CTTTAATAAG GAGATATACC ATGGGCAGCA GCCATCACCA TCATCACCAC M G S S H H H H H H

SacI AscI SbfI SalI NotI BamHI EcoRI EcoICRI BssHII PstI AccI HindIII101AGCCAGGATC CGAATTCGAG CTCGGCGCGC CTGCAGGTCG ACAAGCTTGC S Q D P N S S S A R L Q V D K L A

Design of the Insert

Design of the Insert71 ATGGGCAGCAGCCATCACCATCATCACCAC M G S S H H H H H H SacI AscI SbfI SalI BamHI EcoRI EcoICRI PstI AccI HindIII101AGCCAGGATCCGAATTCGAGCTCGGCGCGCCTGCAGGTCGACAAGCTTGC S Q D P N S S S A R L Q V D K L A

The gene we want:ggctgcgacagggcgagcccgtactgcggttaa G C D R A S P Y C G *

BamHI PstI AGCCAGGATCCGAATTCGAGCTCGGCGCGCCTGCAGGTCGACAAGCTTGC S Q D P N S S S A R L Q V D K L A G C D R A S P Y C G * ggctgcgacagggcgagcccgtactgcggttaa

AGCCAGGATCCGggctgcgacagggcgagcccgtactgcggttaaCTGCAGGTCGACAA

Be aware of the amber stop codon: TAG

Multiple cloning site

Design of the Insert

Always check and re-check your sequence!

ATGGGCAGCA GCCATCACCA TCATCACCACAGCCAGGATCCGggctgcgacagggcgagcccgtactgcggttaaCTGCAGGTCGACAA

atgggcagcagccatcaccatcatcaccacagccaggatccgggctgcgacagggcgagc M G S S H H H H H H S Q D P G C D R A S ccgtactgcggttaactgcaggtcgacaa P Y C G - L Q V D

Everything looks good: in frame the whole way!

Translate the whole gene

The wrong way to do it:AGCCAGGATCC ggctgcgacagggcgagcccgtactgcggttaaCTGCAGGTCGACAAGCTT

atgggcagcagccatcaccatcatcaccacagccaggatccggctgcgacagggcgagccM G S S H H H H H H S Q D P A A T G R A cgtactgcggttaactgcaggtcgacaagcttR T A V N C R S T S

Frame shifted = garbage!

Design of the Insert

The gene is just inserted after the restriction site, which is out of frame with the plasmid-encoded start-codon/His-tag

**Some plasmids, for whatever reason, have restriction sites out of frame with the translated

gene**

Finishing Touches

atgggcagcagccatcaccatcatcaccacagccaggatccgggctgcgacagggcgagc M G S S H H H H H H S Q D P G C D R A S ccgtactgcggttaactgcaggtcgacaa P Y C G - L Q V D

•Restriction enzymes need 5’ and 3’ base pairs to cut properly

•NEB has a reference guide for specific enzymes (see link below)

•A good rule of thumb is 6 base pairs after the recognition site

•Inserting a GC “clamp” at the end and beginning of the sequence is also a good idea

http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/cleavage_linearized_vector.asp

gccagccaggatccgggctgcgacagggcgagcccgtactgcggttaactgcaggtcgacgc S Q D P G C D R A S P Y C G - L Q V D

Final gene, polished and ready to go:

Once the insert is designed correctly, the next step is designing primers to order from IDT, based on insert synthesis strategy

Design of the Primers

Three main strategies towards insert synthesis:

•PCR amplification

•Klenow extension of overlapping primers

•Complimentary full-length primers

+Insert

Vector

The most common method of insert synthesis

•Necessitates a pre-existing construct

•Extra restriction sites and/or amino acid residues can be added on each side of the gene

•Internal mutations are more difficult

PCR Amplification of Insert from an Existing Gene

Insert

PCR amplification from overlapping primers

•No pre-existing construct is needed

•PCR products messy, possibly making subsequent rxns difficult

•Good for inserts >150 bp

PCR Synthesis of Insert

F1: 10xF2: 1x

R1: 1xR2: 10x

5’3’

5’ 3’

5’3’

5’ 3’

Full-length insert should still be the major productInsert

Klenow Extension of Overlapping Primers

•Two primers that are complimentary in their 3’ region are designed (overlap 15bp)

•Extended to full length by the Klenow fragment of DNA Polymerase I

•Useful if insert is 50 to 150 bp

Insert

5’3’5’ 3’

5’3’

5’ 3’

KlenowKlenow fragment: retains 3’ to 5’ polymerase activity, but does not have exonuclease activity

•The simplest approach

•Order two primers that compliment each other

•Mix the two primers, heat, and aneal slowly (to ensure proper base-pairing)

•Feasible if the total insert size is < 60 bp

Complimentary Full-Length Primers

Insert5’3’

5’ 3’ Anneal

Designing Primers to Order

Once the insert synthesis technique is decided, primer design is fairly straight-forward

Forward primers:

•Assess necessary overlap and copy the sequence from your designed gene, along with extra 5’ sequence

Reverse primers:

•First, design exactly as if it were a forward primer: Copy necessary overlap and extra 3’ sequence from your designed gene

•Once all this is in place, use pDRAW32 sequence manipulator to calculate the reverse compliment

•Order the pDRAW32 calculated sequence directly

Cloning Out an Existing GeneIn the example mentioned previously, we would normally use full length overlapping primers, but let’s look at the more common case of having a preexisting gene:

gccagccaggatccgggctgcgacagggcgagcccgtactgcggttaactgcaggtcgacgc S Q D P G C D R A S P Y C G - L Q V D

tgcggcccagccggccatgggctgcgacagggcgagcccgtactgcggtggaggcggtgctgcagcgc A A Q P A M G C D R A S P Y C G G G G A A A

Preexisting gene:

Goal gene:

gccagccaggatccgggctgcgacagg ccgtactgcggttaactgcaggtcgacgc

Forward Primer: Design of Reverse Primer:

+

Overlap

Extra sequence from gene design

gccagccaggatccgggctgcgacagggcgagcccgtactgcggttaactgcaggtcgacgc S Q D P G C D R A S P Y C G - L Q V D

Ordering Primers

Forward primer to order:gccagccaggatccgggctgcgacagg

Reverse primer to order:GCGTCGACCTGCAGTTAACCGCAGTACGG

http://www.idtdna.com/Home/Home.aspxNow we can order the primers:

Design of Reverse Primer: ccgtactgcggttaactgcaggtcgacgc

&

Vectors and Bacteria Strains

Vector Promoter E Coli strains we use

pQE-30 T5 promoter XL1-Blue: mostly good for DNA isolation/phage displayM15(pREP4): tighter regulation of the lac suppressor

pMAL Ptac promoter

pCANTAB-5E Plac promoter

pET-DuetpRSF-Duet

T7 lac promoter(An E. Coli strain with phage T7 RNA polymerase is necessary)

BL-21: Protease deficient, stable to toxic proteins, and contains the T7 RNA polymerase gene

An important thing to think about before you start cloning: What vectors/E Coli should I use?

lac sitePromoter RBS ATG- your gene

lac repressor lac

sitePromoter RBS ATG- your gene

RNA polymerase

X

IPTG (or lactose, etc)

IPTG

lac sitePromoter RBS ATG- your gene

Transcription

mRNA

lac Expression Regulation

•Anti-biotic resistance (working concentration)

•Ampicillin (100g/mL)

•Kanamycin (35g/mL)

•Tetracycline HCl (10g/mL)

•Chloramphenicol (170g/mL in ethanol)

Purification Tags and Selection (Anti-biotic Resistance)

•Purification Tag

•His-tag (nickel agarose resin)

•Maltose Binding Protein (amylose resin)

•Glutathione S-Transferase (glutathione resin)

Digestion of Insert and Vector

•Digest with the same restriction endonucleases

•Optional (recommended) step:

•Treat the plasmid DNA with Antarctic phosphatase

•Decreases the background by stopping self-ligation of singly cut plasmid and background re-ligation

Ligation of the Insert into the Vector

+

•Ligation covalently attaches the vector and the insert via a phosphodiester bond (5’phosphate and 3’ hydroxyl of the next base)

Antarctic Phosphatase and Ligation

http://www.neb.com/nebecomm/products/productM0202.asp

O

P

O

O O

R1O

OH

O

P

O

O O

R2OO

P

O

O O+

O

P

O

O O

R1O

O

O

P

O

O O

R2OO

PO O

•Antarctic Phosphatase cleaves this phosphate, disallowing self-ligation

•The insert still has the 5’ phosphate though

Transformation

•The functional construct is now ready to be transformed into new E. Coli and grown up

•The new DNA isolated from the E. Coli must then be sequenced to make sure that everything worked

•Once the sequence is confirmed, we are ready to go!