Summary of Two-Hybrid Screening Service
The Screening Core provides a yeast two-hybrid screening service using a LexA-based two-hybrid system. The core will screen one of our libraries or a library provided by a customer to identify proteins that interact with a protein of interest. Customers construct their own "bait" plasmid by inserting their open reading frame into a LexA vector plasmid that we provide. Our laboratory will transform yeast with the bait plasmid and perform background testing to establish the screening feasibility. If the background is sufficiently low, we will screen a library to identify cDNAs encoding proteins that interact with the customer's protein. For each interacting clone we will confirm the two-hybird interaction with the protein of interest, and we will test specificity by assaying for interactions with several unrelated proteins. At the end of a successful screen, we provide customers with the insert sizes of the interacting clones, their interaction profiles, 5'-end sequences, and yeast containing the library plasmid (which can be isolated for further analysis). Details of each step are outlined below. For more information, please contact (info(at)twohybridservice.org).
Open flowchart
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Construction of the Bait Plasmid
We will send you one of the bait vectors that we use for expressing LexA fusions, (pEG202, pNLex, or pHZ5). Maps and sequence files for these vectors can be found here. It is up to you to clone your ORF into the bait vector, in-frame with the lexA ORF. The vectors have a multiple cloning site downstream of the LexA gene. We recommend that your ORF start at the second codon (not the ATG) and continue to include the natural stop codon. Any internal part of an ORF can also be used, but a stop codon should be added to the fragment inserted into the bait vector. Your ORF can be inserted into the bait vector using standard "cut-and-paste" cloning, in vivo recombination cloning, or in vitro recombination (a GatewayTM destination version of the bait vectors are also available upon request). Once you have cloned your ORF, send the resulting plasmid back to us.
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Transactivation Potential
Upon receiving your plasmid, we will transform yeast with it and then determine the background transactivation potential of your bait. This is a measure of whether your protein will activate transcription on its own when expressed as a LexA-hybrid. If the background transactivation is low enough (less than about one in 105 transformants giving rise to a colony on selective medium) we will be able to proceed to the library screening. All steps up to this point are done free of charge.
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Libraries
The two-hybrid screening service includes screening one of the libraries available in the MPC lab. A list of the currently available libraries can be found here. If an appropriate library is not available from us, customers may provide us with DNA for a different library. We will transform yeast with the desired library and perform the two-hybrid screen. There is an additional charge (service B) for use of the customer's library DNA, due to the additional time and materials involved in a large-scale, high-efficiency yeast transformation. Libraries provided to us by customers must be compatible with the LexA yeast two-hybrid system and can be constructed using the library vector that we provide (pJZ4).
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Library screen to identify initial positive interactors
We conduct library screens using our mating protocol. We select up to ~200 potential positives and conduct an initial test to ensure that the expression of the selectable marker is due to transcriptional activation by your fusion protein.
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Confirm two-hybrid interaction and test specificity
We confirm the two-hybrid interaction for each clone by isolating the cDNA insert, re-cloning it into the AD vector, and testing for interaction with the bait protein. We also test for interaction with several unrelated proteins to ensure that the interaction is specific for the original bait protein.
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Sequencing
For confirmed positives we will obtain a 5' end sequence tag to determine the identity of the cDNA. The results of the sequencing will be emailed to you. Sequencing of the top 20 positives is included in the service charge.
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