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High-Throughput Cloning and Expression Library Creation for
Functional Proteomics
The International Proteomics Tutorial Program
What is cloning?
DNA cloningOrganism cloning
Cloning is a process where a genetically identical copy of an organism, cell or DNA is produced
Dolly sheep
DNA cloning and functional proteomics
DNA
Functional proteomics
Cell based assays Cell proliferationMorphologyDrug resistance
Protein functionInteraction partnersPTMs
Gene of interest Clone
Protein expressionin vivo or in vitro
Expression Libraries
Traditional Library DNA Clone Library
Expression library is the collection of clones capable to express protein. It stores genetic information to facilitate the study of the roles of genes and proteins in a systematic and high-throughput (HTP) manner.
Main enzymes used for DNA cloning
(1) DNA polymerases
(2) Restriction enzymes and ligases
(3) Recombinases
DNA polymerase
DNA polymerase can use RNA or DNA as template.
DNA dependent-DNA polymerase copy the DNA and can be used for DNA amplification in vitro in a Polymerase Chain Reaction (PCR)
Polymerase Chain Reaction
DNA polymerase
DNA polymerase can use RNA or DNA as template.
DNA dependent-DNA polymerase copy the DNA and can be used for DNA amplification in vitro in a Polymerase Chain Reaction (PCR)
RNA dependent-DNA polymerase is used to copy the information from the RNA to the DNA. The new DNA molecule is name copy DNA or cDNA
cDNA synthesis
Type II Restriction Enzyme and Ligase
Type II Restriction enzymes – “scissors” capable of identifying and cleaving specific DNA sequences
Ligase - re-join two DNA strands
Recombinases
Recombinases can cut and ligate DNA fragments, within a single complex
Bidirectional recombination
Recombinases
Unidirectional recombination
Recombinases can cut and ligate DNA fragments, within a single complex
Steps for DNA cloning
1) Identification of appropriate insert and insert source
2) Selection of appropriate vector
3) Selection of appropriate cloning strategy
Insert selection - What is the structure of the gene in the genome?
Prokaryotic RNA5’UTR Coding sequence(CDS)Start Stop 3’UTR
Open Reading Frame (ORF)
Prokaryotic genomic DNACoding sequence(CDS)
Transcription
Eukaryotic genomic DNA
Mature eukaryotic mRNA
Pre-mRNA
5’ 3’
Cap 5’UTR Coding sequence(CDS) 3’UTR Poly-A tailStart Stop
3’
Capping, splicing, polyadenylation
Open Reading Frame (ORF)
5’
5’UTR 3’UTRExon ExonExon
Intron IntronIntron
Exon
5’
5’UTR 3’UTRExon ExonExon
Intron IntronIntron
Exon
Insert selection - What is the structure of the gene in the genome?
ORFs – The insert of choice for expression clones
Due to the lack of introns, ORFs can be expressed to study protein functions.
Templates to Generate ORF Clones
(1) Genomic DNA: all genes equally present but not suitable for genes with introns (most eukaryotes);
(2) cDNA: no introns between exons but the biases on mRNA expression exist both temporary and spatially;
(3) Vector with the gene of interest (GOI): gene specific primers can be designed to amplify the GOI for the cloning into the desired vectors;
(4) Gene Synthesis (Chemical or PCR-based method). Allows the codon usage optimization. High cost.
Templates to generate
ORF clones
Vectors
Entry vector common features: • ori (origin of replication)• selection marker • cloning site
Inserts cloned into entry vectors are NOT expressed
Expression vectors
Additional features in the expression vector : • promoter (required) • tag (optional)
Necessary characteristics of high-throughput cloning systems
• Properties of the enzymes used for cloning
• Fidelity
• Efficiency
• Stability
• Clone validation
• Automation Friendly
• Cost
Example of available high-throughput cloning systems
1. Restriction Enzyme Based Cloning(1) Flexi® Vector Systems (Promega)
2. Recombination Based Cloning(1) Creator Cloning System (Cre-loxP, Clontech)
(2) Gateway® Technology (phage lambda – attB/P, Life
Technologies)
Flexi® vector systems• Flexi cloning system is a restriction enzyme-based cloning system that uses two rare-cutting restriction enzymes, SgfI and PmeI
• Flexi vectors carry a lethal gene in between SgfI and PmeI to allow the efficient cloning selection.
Frequency of cDNAs or open reading frames(ORFs) lacking SgfI or PmeI sites
Flexi® Vector Systems Technical Manual@Promega
Creating Flexi® clones
Lethal gene in the expression vectors provides negative selections for the parent vectors
Generation of Flexi clones with amino-terminal tag
5’…GTTTAAAC…3’3’…CAAATTTG…5’
PmeI sequence:PmeI sequence:
•Reconstitution of SgfI site encodes Ala-Ile-Ala and PmeI site encodes Val-Stop-Thr.• GOI will be translated from the tag’s start codon to the stop codon present in the gene or the PmeI site (in red);
SgfI sequence:SgfI sequence:
5’…GCGATCGC…3’3’…CGCTAGCG…5’
+Digestion,
ligation and
selection
5’…GTTTAAAC…3’3’…CAAATTTG…5’
5’…GAGCTC…3’3’…CTCGAG…5’
PmeI PmeI EcolCRIEcolCRI
5’…GTTTCTCNN…3’3’…CAAAGAGNN…5’
New SequenceNew Sequence
Ligation
• Ligation of PmeI and EcolCRI sites disrupt both PmeI and EcolCRI sites, new sequence encodes Val-Phe-X, X determined by GNN;• It is inadvisable to transfer GOI into C-terminus Flexi vectors if you plan to transfer it into other Flexi vectors in the future.
Generation of Flexi clones with carboxyl-terminal tag
+Digestion,
ligation and
selection
Transferring genes between distinct Flexi vectors Acceptor vector with amino-terminal tag
SgfI & PmeI digestion
LigationTransformation
Antibiotic 2 selection
SgfI & PmeI digestion
SgfI & EcolCRI digestion
LigationTransformation
Antibiotic 2 selection
Transferring genes between distinct Flexi vectors Acceptor vector with carboxyl-terminal tag
+ +
Creator cloning system
•Creating Master Clones With In-Fusion: In-Fusion enzyme fuses DNA fragments, e.g., PCR generated sequences and linearized vectors, precisely by recognizing a 15bp overlap at their ends.
•Creating Expression Clones With Creator Cloning: 1. Cre recombinase mediates the recombination between DNA sequences at loxP sites; 2. Donor vectors contain two loxP sites, which flank the 5’ of GOI and 5’ of CmR resistance gene; 3. Acceptor vectors contain a single loxP site, followed by a bacteria promoter to facilitate the expression CmR gene after recombination.
LoxP sequences:LoxP sequences:
In-Fusion® HD Cloning Kit User Manual@Clontech
Creating entry clones with In-Fusion
Creating expression clones with Creator cloning system
Tags in the Creator expression vectorsN-terminal Tag
Tags in the Creator expression vectorsC-terminal Tag
Gateway cloning system
•Creating Gateway Master Clones Using BP reactions BP clonase recombine linear PCR product with entry vectors.
•Creating Gateway Expression Clones Using LR reactions LR clonase recombine entry clone with expression vectors.
5’UTR 3’UTR
Gene of Interest(GOI)
Generation of PCR fragment with attB sites
Gene of Interest(GOI)
1st PCR using gene specific primers + Partial attB1/attB2 site sequences
2nd PCR using universal primers including the full length attB1/attB2 site sequences
attB1 attB2
Gene of Interest(GOI)
•attB1/attB2 site sequences are adapted from phage λ that allows the site specific recombination between DNA fragments;•Two rounds of PCR are applied for the addition of adaptor sequences to avoid potential mutations in the synthesis of long primers.
Creating Gateway entry clones using BP reactions
Creating Gateway expression clones using LR reactions
Tags in the Gateway expression vectorsN-terminal Tag
Tags in the Gateway expression vectorsC-terminal Tag
Generating new expression vectors
Flexi Cassette
Gateway Cassette
Creator Cassette
Overall principle: Insert the required cloning cassette into the desired expression vectors by any cloning
method (restriction enzyme, In-Fusion).
Alternative cloning methodsIn-Fusion
Alternative cloning methodsGibson Assembly
Alternative cloning methodsLigation Independent Cloning
Alternative cloning methodsGold Gate Assembly
Alternative cloning methodsUnivector Plasmid Fusion System
Comparison of distinct cloning systemsFlexi Creator Gateway
Cloning steps
8 s teps (PCR, digestion, DNA purifi cation, anneal ing,
transformation, colony picking, DNA extraction, sequencing)
12 s teps (PCR, vector digestion, DNA purifi cation/cloning enhance, In-Fus ion, transformation, colony
picking, DNA extraction, sequencing, Cre-reaction,
transformation, colony picking, DNA extraction, clone va l idation )
12 s teps (PCR1+PCR2 - s ingle tube reaction, DNA purifi cation, BP
reaction, transformation, colony picking, DNA extraction, sequencing, LR-reaction,
transformation, colony picking, DNA extraction, clone va l idation )
Dependent
If BP reaction is employed the gene size should be <3Kb. Genes larger than
3Kb can be cloned using other approaches
Dependent
Genes with either SgfI or PmeI sites should be cloned using alternative
protocols
Necessary Necessary
Alternative: use lox P site (34 nt) as adapter sequence in the PCR reaction
Alternatives: use attL site (100 nt) as adapter sequence in the PCR reaction; or a expression vector with attP sites, which will add 100 nt on each side of
the ORF
Primer lengh (adapter sequence only)
8 nt (restriction enzyme recognition s i te)
15 nt (vector extremity sequence) or 34nt (loxP sequence; cloning
di rectly to the express ion vector)
25 nt (attB s i te) or 42nt (attB s i te + Shine-Dalgarno sequence + Kozak sequence) for the express ion of
native protein
Independent
Size of cloned genes Any Any
Sequence of the gene of interest
Independent
Entry vector Not necessary
Flexi Creator Gateway
N-terminal : 3 N-terminal : 12 N-terminal : 9
C-terminal : 1C-terminal : 0
(loxP s i te i s removed by spl icing) C-terminal : 9
Amino-terminal tags (closed ORFs)
Express ion of the SgfI s i te. Addition of 3 aa between the tag
and the GOI
Express ion of the loxP s i te. Addition of 12 aa between the tag
and the GOI.
Express ion of the attB1 s i te. Addition of 9 aa between the tag
and the GOI.
Carboxyl-terminal tags (native start codon)
Express ion of the EcoICRI s i te. Addition of 3 aa between the tag
and the GOI
Express ion of C-termina l tag from the express ion vector only i f the
express ion systems is able to perform RNA spl icing
Express ion of the attB2 s i te. Addition of 9 aa between the tag
and the GOI
Ligation – Medium In-Fus ion – LowBP reaction – Low (<3Kb genes),
High for larger genes
Cre reaction - High LR reaction – Low
Bacterial competency needed (cfu)
10^6 10^8 10^6
Number of expression vectors compatible with
the system
+ ++ ++++
Number of clones libraries compatible with the
system
+ ++ ++++
Cost per construct (enzymes only)
$4.80 - $21.02 (depending on the vendor)
$14.00 - $28.10 (In-Fus ion - $12.98, Cre - $1.02-
15.12)
$16.33 (BP mix - $7.99, LR mix - $8.34)
Potential number of additional amino acids
introduced via the cloning site
Number of false positives
Proteomics application of expression clone libraries
Generation of protein microarray with 2000 distinct ORFs from Vibrio cholerae for
the identification of immune response in patients with Cholera
Adapted from Festa et al, 2012
DNA
Protein