Removing the Restrictions from Molecular Cloning
Ben Borgo, Ph.D., M.B.A. Global Product Manager
For Research Use Only. Not for use in diagnostic procedures.
•
•
Expertise in Oligo Library Synthesis & Precision Instruments -
Market leaders in microarrays and scanners Market leaders in NGS target enrichment (SureSelect & HaloPlex) Market leading Bioanalyzers and Workflow Automation Solutions
-
Market leading Mass Spec and LC-MS
Custom Manufacturing of Oligonucleotides -
Cost-effective customization of customer designs Microarray fabrication facility offers ability to manufacture oligonucleotides in a massively parallel way independent of sequence. High fidelity, longest oligos commercially available
-
•
•
Enzyme and Protein Engineering Capabilities -
Specialty polymerase enzymes developed with licensed directed evolution technology can be leveraged in specialty applications
-
Mutagenesis methods for developing proteins with programmed genetic changes Cloning and protein expression products
Creating a New Legacy in Synthetic Biology -
Ongoing collaborations with leading synthetic biology institutions Several new and up coming product offerings •
QuikChange HT protein engineering kit
•
SureGuide CRISPR/Cas9 Nuclease kit
•
SureVector Next-generation Cloning
2 For Research Use Only. Not for use in diagnostic procedures.
Technological Leaders in Cloning at Agilent - Agilent’s molecular cloning portfolio includes over 30 unique vectors for cloning and expression in bacterial, yeast and mammalian systems. AdEasy
Specialty Vectors
Competent Cells
StrataClone
New wave of synthetic biology methods is encouraging scientists to explore the next generation offerings rather than sticking with traditional RE cloning
For Research Use Only. Not for use in diagnostic procedures.
Traditional RE-based Cloning Select vector backbone PCR and digest GOI – 2 hours Gel purify – ½ hour Digest vector backbone – 2 hours
Gel purify – ½ hour Adgene.org
Combine and ligate – ½ hour Choose N or C-terminal tag, promoter, origin Transform and screen Make sure correct RE present in MCS Make sure RE sites in MCS have sufficient separation REs used can’t be present in sequence of GOI
Common cause of failure: inefficient cleavage of insert, vector or both For Research Use Only. Not for use in diagnostic procedures.
Next-Gen Cloning with Gibson Assembly/Overlap Based Methods Overlapping, homologous ends Select vector backbone PCR GOI – 2 hours Digest vector backbone – 2 hours
Multiple enzymes prep, combine segments in 1-step
Gel purify – ½ hour Combine and assemble – ½ hour Transform and screen
Completed construct For Research Use Only. Not for use in diagnostic procedures.
Modular Vector Assembly - Never need to use a linearized vector
- Each component is a substitutable fragment - Extensive set of standard parts - Amenable to automation
For Research Use Only. Not for use in diagnostic procedures.
Assembly Protocol I. Standard parts: DNA fragments designed for orthogonal assembly XP1 Fragment
Selectable Marker
End User GOI Promoter-Tag
Origin of Replication
XP2 Fragment
II. Seamless Assembly
Next-gen Cloning Enzyme Mix Reaction 95%
60-80%
>95%
Number of fragments
Up to 6
Up to 15
Up to 6
Up to 6
Up to 4
7+
Number of vectors available
4400
4400
4400
4400
4400
1,000,000+
Simultaneous assembly of multiple constructs
No
No
No
No
Yes
Yes
Swappable elements
No
No
No
No
No
Yes
Agilent Confidential
For Research Use Only. Not for use in diagnostic procedures.
Element Swapping with SureVector - Fully assembled SureVector construct - Replace GOI with another GOI - LacZ control fragment, produces blue colonies when plated on media containing Xgal - Plasmid containing septin gives white colonies T7-His
* Too many to count
*
600
*
500 400 300
blue white
200 100
LacZ
0
septin
Septin -> LacZ (Dpn+)
Septin -> LacZ (Dpn-)
Septin -> Septin (Dpn+)
Septin -> Septin (Dpn-)
KanR
p15A
- DpnI digest required to remove excess parental plasmid - 87% efficiency with DpnI digest For Research Use Only. Not for use in diagnostic procedures.
Element Swapping with SureVector - Fully assembled SureVector construct p15A-XP1-LacI-T7-His-Kan - Replace AB selection marker with Amp or Kan - Plate on desired media and sequence T7-His
160 140 120 100 80 60 40
AmpR/ ClrR
LacZ
180
20 0
Kan -> Amp (Dpn+)
Kan -> Kan -> Clr Kan -> Clr Kan -> Kan Kan -> Kan Amp (Dpn-) (Dpn+) (Dpn-) (Dpn+) (Dpn-)
KanR
p15A
- Both Kan->Amp and Kan->Clr successfully swapped - DpnI optional, as AB selection is strong enough to only get correct colonies - 8 of 8 colonies sequenced for both amp and clr showed the swapped fragment was present
For Research Use Only. Not for use in diagnostic procedures.
Multifragment Assembly with SureVector
- 3 component assembly - Three origins (pUC, p15A, pBR322) in one assembly - Kan-XP1-LacI-His6-PTac-LacZ (LacZnegative control) - 24 colonies screened, 2 undetermined - Low background, high CFU ratio for all constructs For Research Use Only. Not for use in diagnostic procedures.
80
CFU’s
70 60 50
40
Blue (LacZ+)
30
White (LacZ-)
20 10 0 Amp-
Kan-
Clr-
Amp
Kan
Clr
120 100
CFU’s
- 3 component assembly - Three selection markers (Amp,Kan,Clr) in one assembly - p15A-XP1-LacI-His6-PTac-LacZ (LacZ- negative control) - Plated on 3 selection media - Low background, high CFU ratio for all constructs
80 60 40
2
4
11
7
p15A-
pUC
pBR322
20 0 LacZ-
LacZ+
Multifragment Assembly with SureVector 14
12
CFU’s
20
10 15
8 6
10
4 5 2 0
0 Amp-
Kan-
Clr-
Amp
Kan
Clr
- Low background, high CFU ratio for all constructs but varied considerable from 5x to 23x - Sequenced 16 colonies from Amp, 16 from Kan and 5 from Clr - 8/9 possible constructs found
For Research Use Only. Not for use in diagnostic procedures.
Colonies correct (sequenced)
- 3x3 component assembly - Three selection markers (Amp,Kan,Clr) + 3 origins (p15A, pUC, pBR322) in one assembly - XP1-LacI-His6-PTac-LacZ (LacZ- negative control) - Plated on 3 selection media
25
CFU ratio p15A pUC19
pBR322
Example Application: Expression Optimization - Protein expression levels vary based on a number of factors, including solubility tag or promoter choice
- Goal: Optimize expression levels for Nedd5 by shuffling components The SureVector Solution: Assemble a small library of vector backbones containing different tags along with a single gene-of-interest Why it’s better: Previously this would require a different vector backbone for each possible tag and a different restriction enzyme/ligation reaction for each tag. With SureVector this can all be done from one kit using a single reaction.
For Research Use Only. Not for use in diagnostic procedures.
SureVector
Traditional Cloning
Other Next-gen Cloning
Speed of protocol
95%
40-70%
60-80%
Assemblies per reaction
10+
1
1
Cost per vector
$80-100
$150-300
$150-300
Number of “catalog” vectors
Over 1 million
~2,500
~2,500
T7
-
We know that different tags or promoters can drastically effect expression levels of a protein But, using traditional techniques we wouldn’t want to build 17 different constructs to screen for expression levels With SureVector all of these constructs can potentially be built using one reaction
-
Tac
-
Rhamnose
-
For Research Use Only. Not for use in diagnostic procedures.
Optimization of Nedd5 expression using a variety of tags All constructs express Nedd5 However, wide range of magnitudes In general, Ptac looks to be a better promoter, with His6 the best tag for high levels of expression Low levels of expression seen with Prhamnose and SBP, or Prhamnose and thioredoxin
SureVector: The world’s first modular vector kit Expansions
Expansions
Core
Yeast and Mammalian Promoter
Yeast and Mammalian Selection
EF1-alpha SV40 CMV CUP1 ADH1 GAL1
Blasticidin Gentamycin Puromycin Hygromycin URA3 HIS3
E. Coli Promoter T7 Tac Rhamnose
Yeast and Mammalian Tags hrGFP HA His FLAG SBP c-Myc
-
Faster than Gibson assembly - 30 min. vs. 60+ min.
-
Largest catalog of vectors available in a single system - Over 1 million configurations -
Most reliable next-gen cloning technique - Validated components means no troubleshooting time
For Research Use Only. Not for use in diagnostic procedures.
E. Coli Tags His GST SBP Thioredoxin HA CBP DsbA MBP
-
Higher efficiency than Gibson - 90+% vs. 60-70%
-
Clone without restriction enzymes
A Complete Cloning Solution
Over 1.2 million possible combinations! For Research Use Only. Not for use in diagnostic procedures.
Vector Construction Software Agilent.com> Genomics > Mutagenesis & Cloning > SureVector Tool -
Explore the possible configurations that can be made with SureVector
-
Export reports and sequence files for SureVector constructs for use in external analysis tools
-
Determine what SureVector kits you’ll need to build a desired vector
-
Order SureVector components using our e-Commerce system
Vector component selection
Vector visualization Agilent Confidential
For Research Use Only. Not for use in diagnostic procedures.
SureVector Select Elements
1) Select components from drop-down menus
2) Upload or paste a gene-of-interest 3) Create to view the map
For Research Use Only. Not for use in diagnostic procedures.
Vector Mapping
Download fastA, GenBank or a PDF report with the vector image
Visual representation of annotated map
For Research Use Only. Not for use in diagnostic procedures.
List of products needed to build this construct
What comes in the kit? -
Core kit (G7514A) -
All DNA fragments shown on the previous page (15)
-
Entry kits -
Select DNA fragments (7)
-
Choose from:
-
SureVector Enzyme Blend & Buffers
•
N-terminal E. coli Promoters (G7518B)
-
XL1-blue Competent Cells
•
C-terminal E. coli Promoters (G7518C)
-
15 reaction kit
•
N-terminal E. coli Tags (G7518D)
•
C-terminal E. coli Tags (G7518E)
•
E. coli Selection markers (G7518A)
-
Expansion kits -
Contains a selection of promoters and tags for: -
E. coli (G7515A,B)
-
Yeast (G7517A,B)
-
Mammalian cells (G7516A,B)
-
15 reactions
For Research Use Only. Not for use in diagnostic procedures.
-
SureVector Enzyme Blend & Buffers 5 reaction kit Coming soon: Purchase only the completed SureVector construct you need or order a custom fragment for any SureVector slot from our gene synthesis partners (Early access for custom fragments available now!)
Fast and Flexible • Less than a day from design to vector • Assemble new vectors as experimental requirements change Easy to Use • Fits cleanly into existing workflows • Single tube assembly method Reliable and Precise Assembly • Extensively tested • Guaranteed to assemble into a functional vector
For Research Use Only. Not for use in diagnostic procedures.
Acknowledgements Peter Sheffield Denise Rhodes Jeffery Bramen Nancy McKinney Sarah Johns Vivian Zhang Rajesh Bagga Yoon Rhee Yvonne Lee Souad Naji
www.agilent.com/genomics/newsurevector
For Research Use Only. Not for use in diagnostic procedures.
Example Application: Optimizing Nedd5 Expression - 5 different N-terminal tags under T7 promoter - GST run on TapeStation failed - HisDbsA and His6 express, DbsA is strongest N-term - CBP, SBP show little to no expression - 6 different C-terminal tags under T7 promoter - 5/6 express, CBP is strongest followed by His6 - HA, Myc show little to no expression
