Controlling the Virulence Pathway Using the Single Clone Riboswitch Sample Clauses

Controlling the Virulence Pathway Using the Single Clone Riboswitch. To In order to more effectively study this signal transduction pathway and infection event this complex system must be simplified. In vivo maximum vir gene induction oc- curs when all three extracellular signals are present; as stated before, some of these sig- nals are synergistically linked to one another. If it were possible to bypass VirA, so that maximum gene induction would occur as the result of just a single extracellular signal, then the system would be easier to study and easier to transport. To achieve this, the sig- nal recognition domains of VirA must be ablated. If only the kinase domain of VirA is expressed with constitutively expressed VirG, then the upregulation of vir genes occurs in absence of any extra cellular signals. We can use this to our advantage by coupling virG expression to riboswitch activity. Controlling virG with the single clone riboswitch discussed in Chapter 2 would create a virulence system that responds only to theophyl- line. VirG would be produced dose-dependently in the presence of varying concentra- tions of theophylline. Any VirG produced would immediately be phosphorylated by the kinase domain of VirA and would upregulate any genes under the control of a vir promo- ter. By fusing a vir promoter, specifically the virB promoter (virBp), to lacZ (virBp::lacZ) we would be able to monitor the effect of theophylline on the VirA/VirG system. To facilitate the transport of the virulence system into E. coli, the 89 C-terminal resi- dues of the -subunit of A. tumefaciens’ RNA polymerase, the RpoA fragment, must be expressed along with the 247 N-terminal residues of E. coli’s XXX xxxxxxxxxx (Xxxxxx, 0000). The wild-type E. coli RNA polymerase is incapable of recognizing and interact- ing with VirG, which is essential for vir gene upregulation (Lorhke, 1999). The RpoA hybrid will be constitutively expressed along with the kinase portion of VirA and theo- phylline dependent VirG in order to create a system that will function in E. coli.
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  • Company to Provide Copy of the Prospectus in Form That May be Downloaded from the Internet If requested by the Representatives, the Company shall cause to be prepared and delivered, at its expense, within one business day from the effective date of this Agreement, to the Representatives an “electronic Prospectus” to be used by the Underwriters in connection with the offering and sale of the Offered Shares. As used herein, the term “electronic Prospectus” means a form of Time of Sale Prospectus, and any amendment or supplement thereto, that meets each of the following conditions: (i) it shall be encoded in an electronic format, satisfactory to the Representatives, that may be transmitted electronically by the Representatives and the other Underwriters to offerees and purchasers of the Offered Shares; (ii) it shall disclose the same information as the paper Time of Sale Prospectus, except to the extent that graphic and image material cannot be disseminated electronically, in which case such graphic and image material shall be replaced in the electronic Prospectus with a fair and accurate narrative description or tabular representation of such material, as appropriate; and (iii) it shall be in or convertible into a paper format or an electronic format, satisfactory to the Representatives, that will allow investors to store and have continuously ready access to the Time of Sale Prospectus at any future time, without charge to investors (other than any fee charged for subscription to the Internet as a whole and for on-line time). The Company hereby confirms that it has included or will include in the Prospectus filed pursuant to XXXXX or otherwise with the Commission and in the Registration Statement at the time it was declared effective an undertaking that, upon receipt of a request by an investor or his or her representative, the Company shall transmit or cause to be transmitted promptly, without charge, a paper copy of the Time of Sale Prospectus.

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