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The Gateway cloning system utilizes what Life Technologies refers to as "Clonase enzyme mix" to catalyze the BP and LR reactions.
What is in this enzyme mix? Is there a sequence for them?
The prototypes for these proteins are:
the bacteriophage λ proteins Int and Xis, required for, respectively the integration and excision of prophages. They are, as mentioned by @WYSIWYG, recombinases.
Genetic map of bacteriophage λ (source of image)
the original Ihf (integrative/integration host factor) was identified as an E. coli protein required for prophage integration.
As you might expect Int and Xis proteins occur in many phage genomes, and in integrated prophages in bacterial genomes, and Ihf, because it is involved in bacterial DNA replication, is ubiquitous. I imagine the actual source of the proteins used by Life Technologies could be a trade secret.
Gateway Recombination Cloning Technology
Invitrogen Gateway recombination cloning uses a one hour reversible recombination reaction, without using restriction enzymes, ligase, subcloning steps, or screening of countless colonies, thereby saving you time, money, and effort. Benefits include:
- 95% cloning efficiency delivers the clone you need
- Maintain orientation and reading frame throughout cloning process
- Efficient cloning of single fragments into multiple vectors simultaneously
- Flexibility to clone multiple gene fragments into a single construct
- Enter the Gateway plaform via Invitrogen TOPO cloning vectors containing Gateway att sites or purchase an Thermo Scientific Ultimate ORF Clone already inserted into a Gateway vector
- New advancements such as MultiSite Gateway Technology make Gateway cloning the ideal cloning method for protein expression and functional analysis.
What are the Gateway clonase enzymes? - Biology
Figure 1 . The Gateway LR in vitro recombination reaction
The Drosophila Gateway™ Vector collection is a set of 68 Gateway-based vectors designed to express epitope-tagged proteins in Drosophila culture cells or flies. At its core is Invitrogen's Gateway™ recombination cassette, which allows you to recombine an Open Reading Frame (ORF) of interest into any of the vectors using a simple and efficient in vitro reaction. The result is a fusion gene with your ORF placed in frame with one of 7 different epitope tags and expressed by one of 4 different promoters.
Gateway™ technology uses lambda integrase to recombine your ORF, flanked by attL1 and attL2 recombination sites, with the attR1 and attR2 recombination sites of a destination vector (figure 1). The result is a "swap" of your ORF with the cassette containing the ccd B gene in the destination vector. Successfully recombined expression clones can be selected based on their resistance to ampicillin and lack of toxicity to standard laboratory strains of E. coli (the ccd B gene product is toxic, which prevents the original destination vector from forming colonies).
Figure 2 . N- and C-terminal Tag/Gateway fusion modules
The Drosophila Gateway Vector collection
We have constructed a set of 17 Gateway/Tag modules (Figure 2 and Table 1) for use in making different destination vectors. Each module contains one of seven epitope tags placed either 5' or 3' of the Gateway cassette, followed by stop codons in all three reading frames. These modules can be subcloned into any vector containing a promoter and terminator of interest as an EcoRV (blunt) - NheI (XbaI compatible) restriction fragment. This subcloning step is facilitated by the presence of a chl R gene within the Gateway cassette, allowing the desired clones to be selected based on their resistance to chloramphenicol.
tag description (abs / em), source
3 FLAG epitopes followed by enterokinase cleavage site, Sigma
tandem FLAG / HA epitopes separated by enterokinase cleavage site
tandem FLAG / Myc epitopes separated by enterokinase cleavage site
Table 1. The 17 Gateway/Tag modules
The 17 modules have two or three letter designations, using combinations of a one-letter abbreviation for each epitope and "W" to stand for the Gateway cassette. Thus, "GW" designates an EGFP tag placed 5' of the Gateway cassette, suitable for producing N-terminal fusions.
GAL4-driven somatic expression in vivo
GAL4-driven somatic and female germline expression in vivo
Table 2 . The four vectors currently available with the 17 Gateway/Tag modules
The 17 modules are currently available in four different vectors (Table 2). The Actin5C- and Hsp70-based vectors (Huynh and Zieler, 1999) are intended for high and moderate levels, respectively, of transient expression in tissue culture cells. The UASt vectors contain a GAL4-responsive promoter for expression in vivo in cells expressing GAL4 (Brand and Perrimon, 1993). The UASp vectors contain a GAL4-responsive promoter modified to allow expression in the female germline in addition to somatic cells (Rorth, 1998), although anecdotal evidence suggests that UASp vectors may have some basal expression in the absence of GAL4 and induce less strongly in response to GAL4 than UASt vectors.
How to get the vectors
It has been a tedious and time-consuming process to arrange for permission to distribute the vectors from the various companies that hold applicable patents (Invitrogen, Sigma, Amersham, Aurora Biosciences, Riken Brain Science Institute, and UCSD/HHMI). Most of the arrangements have been finalized, with the exception of Amersham, which has reneged on their initial approval. It has also been impossible to make any progress with AmershamÕs lawyers due to the pending sale of Amersham to GE. Unfortunately, Amersham has legal control over distribution of GFP-based vectors, precluding us from distributing 24 of the 68 vectors at this time. We are continuing to work on ways around this legal roadblock and are hopeful that this barrier can be resolved, but we have no idea how long these negotiations will take. We also do NOT have blanket permission to distribute the mRFP-based vectors however, interested labs can contact Roger Tsien and sign an individual MTA with HHMI/UCSD after which we can provide the 8 mRFP-based vectors.
As a further complication, we had planned on providing the entire collection of vectors as a microtiter plate of glycerol stocks, which were prepared LONG ago and currently sit in our freezer. Given that many people have been awaiting these vectors for 6 months or more, we can ship DNA of a small number of FLAG-, myc-, HA-, or mRFP-based vectors to not-for-profit institutions upon request. Information on obtaining a Material Transfer Agreement can be found here.
Note that, under the terms of the MTA, you are not allowed to further distribute these vectors to parties that have not also signed an MTA with the Carnegie Institute. New labs and departing post-docs and students that want to take copies of these vectors should fill out their own MTA so that we don't get sued.
Organization of the vectors
Table 3 . Organization of vectors in the microtiter plate
The vectors are organized in a microtiter plate (table 3). All vectors are resistant to ampicillin (in the vector backbone) and chloramphenicol (in the Gateway cassette). Plates that include the mRFP-based vectors will only be sent to labs that have signed a separate MTA with Roger Tsien otherwise column 11 will be omitted. We have taken every precaution to ensure that the proper clones are in each well however, we strongly recommend that you verify the structure of the destination vectors you are using before proceeding with your experiments. Suggestions for enzymes to cut vectors with specific epitopes are in table 4, and the predicted restriction fragment sizes are available in TEXT and JPEG files listed with the sequence files (table 7).
(1) EGFP and ECFP cannot be distinguished from each other except by sequencing, and are also very similar to Venus with the exception of a PstI site in Venus.
(2) No useful cut sites exist in EGFP and ECFP, but can be distinguished based on size relative to the other epitopes.
Table 4 . Restriction enzymes with sites in the listed epitope for use in verifying destination vectors
I strongly recommend thoroughly reading the Gateway™ Technology manual from Invitrogen. The information we've provided here is only intended to demonstrate how we routinely use the Gateway system in our lab.
How do I flank my ORF with attL1 and attL2 recombination sites?
Several options are available to produce an Entry clone with your ORF positioned in the proper reading frame. We routinely amplify the desired ORF and clone it into an Entry vector by a topoisomerase-catalyzed reaction (pENTR/D-TOPO Cloning Kit, Invitrogen catalog # K240020). Primers are designed with the sequence CACC on the 5' end of the 5' primer followed by the gene specific sequence in the first reading frame. The 5' end of the 3' primer should end with a complete codon (Figure 3). If you plan on making C-terminal fusions, your PCR product must include an ATG, typically immediately after the CACC tail, and must not contain a stop codon. Note that the CACC sequence is a consensus Kozak site and will be included in the final clone. The resulting construct can also be used to make N-terminal fusions, but will contain a short tail at the C-terminus which can be detrimental to some proteins.
Figure 3 . PCR product design for TOPO cloning
The resulting Entry clone will have the sequence shown in figure 4, where the AAA-AAA and TAC-AAA sequences represent the core of the attL1 and attL2 recombination sites.
For the PCR reaction, we routinely use a proofreading polymerase such as Pfx (Invitrogen catalog # 11708-021) to amplify from a cDNA, and have observed a PCR-induced error rate of
1 / 15,000 bases after cloning from a 25-cycle amplification. In our hands, we need to use a much lower concentration of MgSO4 in the PCR reaction (0.25 mM) than recommended by Invitrogen in order to get a PCR product.
We have found that the TOPO cloning reaction isn't as efficient or directional as claimed by Invitrogen. We recover a median of 42 colonies (range 17 - 356), of which a median of 25% (range 8 - 56%) contain the PCR product cloned in the correct orientation. However, the colonies can be rapidly screened by colony PCR using a vector specific and ORF specific primer. Afterwards we miniprep and sequence verify a single clone, which becomes our master Entry clone for recombination into the desired Destination vectors.
Entry clones can also be produced by including attB1 and attB2 sequences in the PCR primers and using a BP recombination reaction to recombine the attB sites with attP sites in a donor vector, resulting in an entry clone with your ORF flanked by attL1/attL2 recombination sites. This strategy can also be used to directly recombine your ORF into a Destination vector by sequentially carrying out a BP followed by a LR recombination reaction, as described in the Gateway manual. However, we prefer to use TOPO cloning to avoid ordering primers that include the 29-bp attB1/attB2 sequences. We also prefer sequence verifying the Entry clone which can then be used with many different Destination vectors.
Figure 4 . Entry clone sequence after cloning into pENTR/D-TOPO
How do I ensure that my ORF will be in the proper reading frame after recombining into a destination vector?
As long as your ORF is in frame with the AAA-AAA sequence at the 5' end and the TAC-AAA sequence at the 3' end (e.g., Figure 4), the resulting clone will be in frame with either N-terminal or C-terminal epitope tags.
What materials do I need to use the Drosophila Gateway Vector Collection?
To make an Entry clone containing your ORF using pENTR/D-TOPO:
- pENTR/D-TOPO Cloning Kit (Invitrogen catalog # K240020, 20 reactions, $20 / rxn), which includes TOP10 competent cells
- cDNA of your favorite gene (DGC clone or equivalent, $20 per clone + shipping from BACPAC)
- PCR primers to amplify the ORF (
We occasionally perform half-reactions of the TOPO cloning step to reduce costs, although this results in a 3 ul reaction volume which can be hard to manage.
To recombine an Entry clone with a Destination clone:
- miniprep DNA of an Entry clone containing your ORF
- miniprep DNA of the desired Destination vector
- Gateway LR Clonase Enzyme Mix (Invitrogen catalog # 11791-019, 20 reactions or 11791-043, 100 reactions $12.50 to $14.75 / rxn)
- Library Efficiency DH5alpha competent cells (Invitrogen catalog # 18263-012 or equivalent $2.50 / 20 ul)
- LB-agar plates containing 100 ug/ml ampicillin
We routinely perform half-reactions (10 ul total volume) of the LR Clonase step to reduce costs, and recently had success with quarter-reactions (5 ul total volume).
Which destination vector should I use?
The beauty of this system is that you can easily make several constructs for different purposes. For example, we initially examine protein localization in Drosophila KC cells using pHGW and pHWG (Hsp70 promoter, GFP fusions at either end). This way we can test which end of the protein better tolerates the epitope tag. Proteins that we want to further analyze in vivo are then swapped into pTGW or pTWG. The HA, Myc, and FLAG epitope tags are all useful for immunoprecipitations in culture or in vivo (we have only used the HA tag in tissue culture experiments).
We are just beginning to use the Venus and mRFP constructs. The Venus tag (EYFP) should be more versatile than EGFP -- it is brighter, works with standard GFP filter sets, and can be easily spectrally separated from ECFP for double labeling experiments. Consequently, we anticipate that it will become our main tag for use in transgenic flies. mRFP fluorescence is relatively weak, but it doesn't have the aggregation and toxicity problems of DsRed. We have successfully used it to look at protein localization in culture cells, but we expect that it will be more difficult to detect in flies, making it more practical to use Venus and ECFP for double-labeling experiments. We are currently experimenting with the tandem-dimer RFP and other red fluorescent proteins which may give useful increases in fluorescent intensity, but these are unlikely to be added to the vector collection until early 2004.
Invitrogen sells many other useful destination vectors, although in some cases you may want to engineer your own. For example, they sell a N-terminal GST fusion vector for expression in E. coli (pDEST 15, Invitrogen catalog # 11802-014), but this vector lacks a cleavage site between the GST and your protein. Invitrogen also sells yeast two-hybrid vectors (ProQuest, Invitrogen catalog # 10835-031), but they are low copy number vectors and we have had mixed success in obtaining sufficient expression levels to see an interaction.
I have an older Gateway manual that says I need to linearize the destination vector before in vitro recombination. Is this necessary?
No. Somewhere along the way Invitrogen changed their opinion on whether linearizing or relaxing your plasmids is necessary. The recombination reaction works fine with two supercoiled plasmids.
How can I sequence verify my expression clones?
In our hands the Gateway recombination reaction has been very reliable so we typically don't sequence-verify our expression clones. But before making transgenic flies you may want to sequence across the tag-gene junction to verify that the reading frame is intact. You may find the primers in table 5 and figure 5 useful.
Other Techniques Performed by SimVector
TA Cloning: SimVector makes it easy to design TA cloning experiments. The TA cloning wizard accepts both commercial T-vectors or restriction designed T-vectors and combines them with the modified PCR product. You can then generate a highly accurate recombinant DNA plasmid map.
Restriction Cloning: This cloning technique requires restriction enzymes to cut the vector molecule and the molecule to be cloned. SimVector performs restriction enzyme analysis and allows you to filter, annotate and map the restriction enzymes on the desired sequences.
How to clone using Gateway technology
To better understand the process, we’ll walk through an example experiment where we might use Gateway cloning to generate our desired constructs: lentiviral expression of the human KRAS gene in mammalian cells.
Method A: recombination of an attB-PCR product or plasmid with an attP donor vector. In this case, we would use PCR to add attB sites to either end of the KRAS coding sequence. If you choose this strategy, it’s important to include the proper protein expression elements (ribosome recognition sequences, start codon, stop codons, reading frame considerations, etc). This video demonstrates how to use the Snapgene program to design Gateway plasmids.
Method B: TOPO-cloning of the desired insert into an attL-entry-TOPO vector. TOPO cloning adds short end(s) to facilitate cloning into an attL-containing entry vector.
Method C: Restriction cloning of a restriction enzyme fragment containing the DNA of interest and a attL-entry vector. This fragment is inserted in a multiple cloning site (MCS) of an attL-containing entry vector.
Introduction to Enzymes
Inside your body, thousands of different chemical reactions are occurring every second. Even as you read these words, your body is breaking down nutrients, building new materials, and maintaining your body tissues. All of these critical life processes, and many more, are driven by chemical reactions.
However, many chemical reactions occur too slowly to support life processes. For example, chemical reactions that are absolutely essential in creating the building blocks of DNA would take 78 million years in just water. In your body, this reaction happens in 10 milliseconds. How is this possible? This reaction happens so quickly in your body because of enzymes —protein catalysts that speed up the rate of chemical reactions.
Directions: Watch Learn Biology: Cells - Enzymes for an introduction to how enzymes work.
Enzymes are proteins that act as catalysts. Catalysts increase the rates of chemical reactions. Enzymes catalyze a reaction by lowering the activation energy, which is the amount of energy needed to begin a chemical reaction. All chemical reactions need activation energy to get started. Activation energy is needed to speed up the motion of molecules to increase the frequency and force with which they collide.
Lowering activation energy dramatically affects how quickly a chemical reaction is completed. Enzyme-catalyzed reactions occur extremely fast. Many happen millions of times faster than uncatalyzed reactions.
This graph demonstrates the effect enzymes can have on a chemical reaction.
The blue line on the graph shows the reaction taking place without the enzyme, and the red line shows the reaction with the enzyme. Notice that the activation energy needed for the reaction with the enzyme is much lower than the reaction without the enzyme. This means that the reaction can occur more rapidly.
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What are the Gateway clonase enzymes? - Biology
The gene of interst can be amplified from the original vector using primers containing recombination sites . The PCR product , flanked by the B1 and B2 recombination sites is mixed in vitro with a Donor vector and BP CLONASE Enzyme Mix. Site specific recombination between the attP site on the pDonor and the attB site on the PCR product generates a co-integrate plasmid which is subsequently resolved into two molecules: one contains the DNA segment of interest in the new vector backbone (Entry Clone) flanked by attL recombination sites, and the other contains the ccdB toxic gene and will not amplify.
Copy Rights: GATEWAY flyer
The gene of interest is now in the Entry Clone. This Entry Clone can be used for transfering the gene into a variety of expression vectors suitable for different expression systems. These expression vectors are termed "Destination Vectors". Adding the Destination Vector and LR CLONASE Enzyme Mix will cause site-specific recombination between the attL site on the Entry Clone and the attR sites on the Destination vector to generate a co-integrate molecule which is subsequently resolved into two molecules, one contains the gene of interest in the new vector backbone (Expression Clone), and the other is in the by-product vector, containing the ccdB toxic gene that prevents expression in bacteria.
Development of series of gateway binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant transformation
We developed a new series of binary vectors useful for Gateway cloning to facilitate transgenic experiments in plant biotechnology. The new system, Gateway Binary Vectors (pGWBs) realized efficient cloning, constitutive expression using the cauliflower mosaic virus (CaMV) 35S promoter and the construction of fusion genes by simple clonase reaction with an entry clone. The reporters employable in this system are beta-glucuronidase (GUS), synthetic green fluorescent protein with S65T mutation (sGFP), luciferase (LUC), enhanced yellow fluorescent protein (EYFP), and enhanced cyan fluorescent protein (ECFP). The tags available are 6xHis, FLAG, 3xHA, 4xMyc, 10xMyc, GST, T7-epitope, and tandem affinity purification (TAP). In total, 13 kinds of reporter or tag were arranged and were almost applicable to both N- and C-fusions. The pGWBs could be used for many purposes, such as promoter::reporter analysis, observation of subcellular localization by the expression of proteins fused to a reporter or tag, and analysis of protein-protein interaction by copurification and immunodetection experiments. The pGWBs were constructed with modified pBI101 containing a CaMV35S promoter-driven hygromycin phosphotransferase (HPT) gene as the second selection marker. We also constructed pGWBs with the marker HPT driven by the nopaline synthase promoter. By using the pGWB system, the expression of tagged proteins, and the localization of GFP-fused proteins were easily analyzed. Moreover, tissue-specific and inducible gene expression using a promoter was also monitored with pGWBs. It is expected that, the pGWB system will serve as a powerful tool for plasmid construction in plant research.
Highly efficient one-step scarless protein tagging by type IIS restriction endonuclease-mediated precision cloning
Protein tagging with a wide variety of epitopes and/or fusion partners is used routinely to dissect protein function molecularly. Frequently, the required DNA subcloning is inefficient, especially in cases where multiple constructs are desired for a given protein with unique tags. Additionally, the generated clones have unwanted junction sequences introduced. To add versatile tags into the extracellular domain of the transmembrane protein THSD1, we developed a protein tagging technique that utilizes non-classical type IIS restriction enzymes that recognize non-palindromic DNA sequences and cleave outside of their recognition sites. Our results demonstrate that this method is highly efficient and can precisely fuse any tag into any position of a protein in a scarless manner. Moreover, this method is cost-efficient and adaptable because it uses commercially available type IIS restriction enzymes and is compatible with the traditional cloning system used by many labs. Therefore, precision tagging technology will benefit a number of researchers by providing an alternate method to integrate an array of tags into protein expression constructs.