Gateway Technology Overview

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2. Why Gateway� Technology is advantageous Time savings- You usually need to use several different systems in order to truly understand protein function Convenience- You may want to first produce protein and then move into expression Gateway� Technology is a universal cloning technology that provides a highly efficient and rapid route to functional analysis, protein expression, and cloning/subcloning of DNA segments. Based on the well characterized site-specific recombination system of phage l, Gateway� Technology allows you to transfer DNA segments between different cloning vectors while maintaining orientation and reading frame, effectively replacing the use of restriction endonucleases and ligase. The Gateway� Technology is also a powerful method for high efficiency directional cloning of PCR products.Gateway� Technology is a universal cloning technology that provides a highly efficient and rapid route to functional analysis, protein expression, and cloning/subcloning of DNA segments. Based on the well characterized site-specific recombination system of phage l, Gateway� Technology allows you to transfer DNA segments between different cloning vectors while maintaining orientation and reading frame, effectively replacing the use of restriction endonucleases and ligase. The Gateway� Technology is also a powerful method for high efficiency directional cloning of PCR products.

3. A New Cloning Technology Is Needed New approaches to cloning are needed as more and more sequence information becomes available. The need to clone, and subclone, more genes, faster, to understand gene function is becoming a fact of life no matter what your research objective. The challenge now is one of speed, throughput and downstream expression. Existing methods have their strengths and weaknesses. If you wanted to develop a cloning technology to overcome the limitations inherent to these approaches, what would you require? New approaches to cloning are needed as more and more sequence information becomes available. The need to clone, and subclone, more genes, faster, to understand gene function is becoming a fact of life no matter what your research objective. The challenge now is one of speed, throughput and downstream expression. Existing methods have their strengths and weaknesses. If you wanted to develop a cloning technology to overcome the limitations inherent to these approaches, what would you require?

4. Maximize compatibility and flexibility Minimize planning Maintain reading frame Rapid No restriction enzymes No gel purification No ligations High-throughput Designing a New Approach to Cloning This slide establishes the criteria to be met in a new cloning system. � Maximize compatibility and flexibility� Minimize planning� Maintain reading frame� Eliminate the need for restriction enzyme digestion, gel purification and ligation. � Provide high-throughput compatibility� reactions are simple and robust The requirements are impressive � to meet these demands, we developed the Gateway� Technology. This slide establishes the criteria to be met in a new cloning system. � Maximize compatibility and flexibility� Minimize planning� Maintain reading frame� Eliminate the need for restriction enzyme digestion, gel purification and ligation. � Provide high-throughput compatibility� reactions are simple and robust The requirements are impressive � to meet these demands, we developed the Gateway� Technology.

5. Gateway� Technology The Gateway� Technology Ensures simple, fast, robust reactions Takes advantage of site-specific recombination � 1 hr. reaction eliminates restriction enzyme and ligase cloning, and tedious colony screening Maintains reading frame and orientation for downstream expression Delivers high efficiency - typically >95% desired clones Offers unlimited flexibility - any vector can be made Gateway�-compatible Allows you to transfer genes into a variety of applications This slide summarizes the characteristics of Gateway� Technology. As we go through this presentation, I�ll show you data that supports each of these claims. Several site-specific recombination systems have been described, including Cre/loxP and phage l. We started with Cre/loxP, but found that it had limitations for what we wanted to do. We decided to use phage l. Let�s briefly review lambda recombination in E. coli. This slide summarizes the characteristics of Gateway� Technology. As we go through this presentation, I�ll show you data that supports each of these claims. Several site-specific recombination systems have been described, including Cre/loxP and phage l. We started with Cre/loxP, but found that it had limitations for what we wanted to do. We decided to use phage l. Let�s briefly review lambda recombination in E. coli.

6. Phage l Recombination in E. coli In lambda, the integration site is known as attP, in E. coli the site is attB. The attB site is short, only 25 bp, keep this in mind as it will be important later. The att sites contain the binding sites for the proteins that mediate l recombination. The integration reaction (attB x attP) is mediated by the proteins integrase (Int) and host integration factor (IHF). When integration occurs, two new sites are created, attL and attR, flanking the integrated prophage, with no loss of DNA sequence. All the att sites are alike in that they contain a 15-bp recognition sequence for the recombinase (integrase). The reaction can also go in the opposite direction (excision). When attL x attR recombine (mediated by the proteins integrase, host integration factor and excisionase [Xis]), the lambda -DNA is excised from the E. coli genome, recreating the attB site in E. coli and the attP site in l. Key points: 1. Site-specific recombination mediated by phage lambda recombination proteins. 2. The reaction is specific and directional: attB x attP ? attL x attR. 3. Each reaction is mediated by a different combination of enzymes. How did we modify lambda -recombination to make it a powerful, flexible cloning technology? In lambda, the integration site is known as attP, in E. coli the site is attB. The attB site is short, only 25 bp, keep this in mind as it will be important later. The att sites contain the binding sites for the proteins that mediate l recombination. The integration reaction (attB x attP) is mediated by the proteins integrase (Int) and host integration factor (IHF). When integration occurs, two new sites are created, attL and attR, flanking the integrated prophage, with no loss of DNA sequence. All the att sites are alike in that they contain a 15-bp recognition sequence for the recombinase (integrase). The reaction can also go in the opposite direction (excision). When attL x attR recombine (mediated by the proteins integrase, host integration factor and excisionase [Xis]), the lambda -DNA is excised from the E. coli genome, recreating the attB site in E. coli and the attP site in l. Key points: 1. Site-specific recombination mediated by phage lambda recombination proteins. 2. The reaction is specific and directional: attB x attP ? attL x attR. 3. Each reaction is mediated by a different combination of enzymes. How did we modify lambda -recombination to make it a powerful, flexible cloning technology?

7. Gateway� Technology The Gateway� reactions are in vitro versions of the integration and excision reactions. Your goal is to move your gene (or genes) from one vector backbone to another. This slide also introduces you to Gateway� nomenclature. Let�s first consider the LR Reaction for making expression clones. 1. Your sequence of interest is cloned in an Entry vector that is transcriptionally silent, Kmr, and is flanked by two recombination sites (attL1 and attL2). 2. You want to move this sequence of interest to a Destination vector. It contains all the sequence information required for expression, and is Apr. This plasmid also contains two recombination sites (attR1 and attR2) that flank a gene for negative selection, ccdB. 3. You combine the two plasmid DNAs, each with sequences flanked by recombination sites that do not recombine with each other, but will recombine with sites resident on the other molecule. 4. LR Clonase� is comprised of Int, IHF, and Xis; you are doing an in vitro version of the excision reaction. 5. Att1 and att2 sites confer directionality and specificity for recombination, so that only attL1 will react with attR1, and attL2 with attR2. 6. Two recombination events occur to make the expression clone, one between attL1 and attR1 and the other between attL2 and attR2. 7. The product of these two recombination events is the plasmid construct that you want and a by-product (labeled as the donor vector). The desired plasmid is under two forms of selection: antibiotic resistance and negative selection. Selecting for Apr eliminates the starting vector and the by-product; the presence of the negative selection marker eliminates the destination vector and co-integrate molecules. The Gateway� reactions are in vitro versions of the integration and excision reactions. Your goal is to move your gene (or genes) from one vector backbone to another. This slide also introduces you to Gateway� nomenclature. Let�s first consider the LR Reaction for making expression clones. 1. Your sequence of interest is cloned in an Entry vector that is transcriptionally silent, Kmr, and is flanked by two recombination sites (attL1 and attL2). 2. You want to move this sequence of interest to a Destination vector. It contains all the sequence information required for expression, and is Apr. This plasmid also contains two recombination sites (attR1 and attR2) that flank a gene for negative selection, ccdB. 3. You combine the two plasmid DNAs, each with sequences flanked by recombination sites that do not recombine with each other, but will recombine with sites resident on the other molecule. 4. LR Clonase� is comprised of Int, IHF, and Xis; you are doing an in vitro version of the excision reaction. 5. Att1 and att2 sites confer directionality and specificity for recombination, so that only attL1 will react with attR1, and attL2 with attR2. 6. Two recombination events occur to make the expression clone, one between attL1 and attR1 and the other between attL2 and attR2. 7. The product of these two recombination events is the plasmid construct that you want and a by-product (labeled as the donor vector). The desired plasmid is under two forms of selection: antibiotic resistance and negative selection. Selecting for Apr eliminates the starting vector and the by-product; the presence of the negative selection marker eliminates the destination vector and co-integrate molecules.

8. BP Clonase� II Protocol attB-PCR product or expression clone 1-7 ml Donor vector (pDONR�)�150 ng 1 ml TE Buffer, pH 8.0 to 8 ml Add 2 ml BP Clonase� II Enzyme Mix Mix and incubate for one hour at 25oC. 3. Add Proteinase K solution and incubate for 10 min at 37oC. 4. Transform competent and select on appropriate antibiotic-resistant plates to obtain entry clones. To reinforce how simple the reactions are, here is the protocol. The one-hour reaction automatically subclones the gene of interest from one plasmid backbone to another. Proteinase K digestion is done to improve transformation efficiency. Chemically competent cells (efficiency 108/�g) are routinely used, but electrocompetent cells can also be used for transformation. We�ve also introduced the new BP Clonase� II enzyme mix which combines the enzyme and reaction buffer, reducing the steps involved in setting up your reaction. In addition, Clonase� II enzyme mixes are stable at �20 degrees storage. To reinforce how simple the reactions are, here is the protocol. The one-hour reaction automatically subclones the gene of interest from one plasmid backbone to another. Proteinase K digestion is done to improve transformation efficiency. Chemically competent cells (efficiency 108/�g) are routinely used, but electrocompetent cells can also be used for transformation. We�ve also introduced the new BP Clonase� II enzyme mix which combines the enzyme and reaction buffer, reducing the steps involved in setting up your reaction. In addition, Clonase� II enzyme mixes are stable at �20 degrees storage.

9. LR Clonase� II Protocol Entry Clone 50-150 ng Destination Vector 150 ng TE Buffer, pH 8.0 to 8 ml 1. Add 2 ml LR Clonase� II enzyme mix 2. Mix and incubate for one hour at 25oC 3. Add Proteinase K solution and incubate for 10 min at 37oC 4. Transform competent E. coli and select for the appropriate antibiotic-resistant expression clones You now have an expression clone you can use for transfection, transduction, or other experiments (depending upon the destination vector) To reinforce how simple the reactions are, here is the protocol. The one-hour reaction automatically subclones the gene of interest from one plasmid backbone to another. Proteinase K digestion is done to improve transformation efficiency. Chemically competent cells (efficiency 108/�g) are routinely used, but electrocompetent cells can also be used for transformation. To reinforce how simple the reactions are, here is the protocol. The one-hour reaction automatically subclones the gene of interest from one plasmid backbone to another. Proteinase K digestion is done to improve transformation efficiency. Chemically competent cells (efficiency 108/�g) are routinely used, but electrocompetent cells can also be used for transformation.

10. Optimization of Protein Expression Selection of best host/vector system Selection of the best tag N-terminal C-terminal The real strength of Gateway� Technology is in the construction of expression clones � in many ways, you can think of Gateway� Technology as the best way to get from cloning to protein expression. Protein expression, using conventional methodology, can be frustrating and time consuming. Optimization of protein expression involves selecting the appropriate host/vector system, as well as selecting the best tag and its position (N-terminal or C-terminal fusion) for your particular requirements. Gateway� overcomes the limitations associated with conventional methodology by allowing the construction of multiple expression vectors in parallel. The real strength of Gateway� Technology is in the construction of expression clones � in many ways, you can think of Gateway� Technology as the best way to get from cloning to protein expression. Protein expression, using conventional methodology, can be frustrating and time consuming. Optimization of protein expression involves selecting the appropriate host/vector system, as well as selecting the best tag and its position (N-terminal or C-terminal fusion) for your particular requirements. Gateway� overcomes the limitations associated with conventional methodology by allowing the construction of multiple expression vectors in parallel.

11. Recombinant Protein Expression Use this as a guide to help you decide which host systems to use for your protein(s) of interest. As an example, you can start with an E. coli-based system if you�re looking for ease of use and high yields of protein. However, as you will see in subsequent slides, testing multiple systems in parallel will allow you to quickly determine which system offers the greatest benefits for your research needs. Use this as a guide to help you decide which host systems to use for your protein(s) of interest. As an example, you can start with an E. coli-based system if you�re looking for ease of use and high yields of protein. However, as you will see in subsequent slides, testing multiple systems in parallel will allow you to quickly determine which system offers the greatest benefits for your research needs.

12. Destination Vectors A number of different destination vectors have been constructed. Destination vectors are expression vectors that have the ccdB gene cloned between the attR sites. They have been designed to support the expression of native, N-terminal and C-terminal fusion proteins in a wide variety of prokaryotic and eukaryotic systems. For an updated list, please refer to the main page at www.invitrogen.com/gateway. A number of different destination vectors have been constructed. Destination vectors are expression vectors that have the ccdB gene cloned between the attR sites. They have been designed to support the expression of native, N-terminal and C-terminal fusion proteins in a wide variety of prokaryotic and eukaryotic systems. For an updated list, please refer to the main page at www.invitrogen.com/gateway.

13. Vector Conversion You can also convert your favorite vector to the Gateway� platform with the Gateway� Vector Conversion System. Following a simple, blunt-end ligation with the Gateway� cassette, you effectively replace your standard polylinker and restriction sites with the att R sites necessary for the recombination event. In addition to the att R1 and att R2 sites, you�ll also introduce a chloramphenicol (Cm) resistance marker for selection of the vector in E. coli. The ccdB gene also allows for the highly efficient selection of expression clones after the LR reaction.You can also convert your favorite vector to the Gateway� platform with the Gateway� Vector Conversion System. Following a simple, blunt-end ligation with the Gateway� cassette, you effectively replace your standard polylinker and restriction sites with the att R sites necessary for the recombination event. In addition to the att R1 and att R2 sites, you�ll also introduce a chloramphenicol (Cm) resistance marker for selection of the vector in E. coli. The ccdB gene also allows for the highly efficient selection of expression clones after the LR reaction.

14. Subcloning an Entry Clone into Multiple Destination Vectors Given the simplicity of the reaction, you can readily visualize numerous DNA sequences being transferred into a variety of downstream applications. For further information, refer back to our Gateway� mini series for various expression systems including our newest RNAi and cell-free applications. Given the simplicity of the reaction, you can readily visualize numerous DNA sequences being transferred into a variety of downstream applications. For further information, refer back to our Gateway� mini series for various expression systems including our newest RNAi and cell-free applications.

15. Top Ten Reasons to Start Using Gateway� Technology Proven technology with over 150 global scientific publications Open Architecture promotes rapid scientific advancement and collaborations with nonrestrictive licensing Fast and simple�you can start using Gateway� Technology in 5 minutes with the new TOPO� TA cloning vector Easier and more cost effective with new Clonase� II Enzyme Mixes Efficient and robust�shuttle to a variety of systems and applications with >95% efficiency every time A variety of applications are already part of the Gateway� platform for expression, solubility, detection, purification, and knockdown studies Flexible�easily convert your favorite vectors to the Gateway� platform Free web-based tools to aid in primer design, in silico cloning, and ordering Expansive collection of Ultimate� ORF Clones ready for immediate use Dedicated resources to ensure and improve the highest level of service and innovation for the Gateway� Technology

16. This is your slide for seminar titles and acknowledgements.This is your slide for seminar titles and acknowledgements.

17. Gateway� Clone Distributors Open BioSystems � U.S.A. RZPD � Germany MRC � United Kingdom John Innes Genome Center � U.S.A. Arabidopsis Biological Resource Center (affiliated with Ohio State University) � U.S.A. Arizona Genome Institute (affiliated with University of Arizona) � U.S.A. Please check back as we�re continually updating our agreements

18. This is your slide for seminar titles and acknowledgements.This is your slide for seminar titles and acknowledgements.

19. What are the new developments? New Distribution policies Academic and government entities can make, sell, and distribute Gateway� entry and expression clones For-profit entities may distribute Gateway� entry and expression clones