Control of the expression of anchored genes using micron scale heaters

Abstract

The present invention demonstrates that gene expression can be controlled in vitro using DNA (gene) sequences immobilized on a template with micron scale temperature heaters. Such expression is controllable by varying temperature of the template on a short time scale. The present invention further demonstrates that nucleic acid constructs controlled by the present method express protein either free or bound to the nucleic acid. Based on these findings, the present invention provides methods and apparatuses useful for the preparation of in vitro programmable protein networks and protein micro arrays.

What is claimed is:  
     
         1 . An in vitro method of controlling gene expression comprising immobilizing at least one nucleic acid construct on a temperature-controlled template, applying a cell extract and expressing at least one protein.  
     
     
         2 . The method of  claim 1  further comprising detecting the expressed protein.  
     
     
         3 . The method of  claim 1  wherein said nucleic acid construct comprises a control region, a coding sequence and spacer region.  
     
     
         4 . The method of  claim 3  wherein said control region comprises a promoter sequence.  
     
     
         5 . The method of  claim 3  wherein said coding sequence comprises a polymerase chain reaction (PCR) deleted stop codon.  
     
     
         6 . The method of  claim 3  wherein said spacer region comprises at least about 70 base pairs.  
     
     
         7 . The method of  claim 4  wherein said promoter sequence comprises a T7 promoter.  
     
     
         8 . The method of  claim 1  wherein said nucleic acid construct is DNA.  
     
     
         9 . The method of  claim 8  wherein said DNA construct is C-terminally biotinylated.  
     
     
         10 . The method of  claim 1  wherein said temperature-controlled template comprises one or more metal oxide pads affixed to a substrate.  
     
     
         11 . The method of  claim 10 , wherein said metal oxide pads further comprise indium tin oxide (ITO).  
     
     
         12 . The method of  claim 10  wherein said substrate is glass.  
     
     
         13 . The method of  claim 10  wherein said metal oxide pads contain avidin-coated beads.  
     
     
         14 . The method of  claim 10  wherein said metal oxide pads comprise about 10 um by about 10 um to about 100 um by about 100 uM.  
     
     
         15 . The method of  claim 1  wherein said template is controlled at a temperature of about 5° C. to about 100° C.  
     
     
         16 . The method of  claim 10  wherein said metal oxide pads have a resistance of about 50 Ohms.  
     
     
         17 . The method of  claim 1  wherein said template is heated by means of electrodes attached to a metal oxide pad.  
     
     
         18 . The method of  claim 17  wherein said electrodes conduct an applied dc current.  
     
     
         19 . The method of  claim 18  wherein said dc current applied is about 1 mA to about 40 mA.  
     
     
         20 . The method of  claim 10  wherein said metal oxide-substrate is mounted on a water-cooled brass sample plate.  
     
     
         21 . The method of  claim 20  wherein said water-cooled brass sample plate is maintained at a temperature wherein protein expression is prevented.  
     
     
         22 . The method of  claim 21  wherein said water-cooled brass sample plate is maintained at about 5° C.  
     
     
         23 . The method of  claim 17  wherein said template is heated to a temperature which causes an onset of protein expression.  
     
     
         24 . The method of  claim 23  wherein said protein expression is stopped by reducing the heat applied to said template.  
     
     
         25 . The method of  claim 1  wherein said expressed protein is localized with the immobilized nucleic acid.  
     
     
         26 . The method of  claim 1  wherein said expressed protein is released from the immobilized nucleic acid.  
     
     
         27 . An apparatus for controlling gene expression comprising a temperature-controlled template having a nucleic acid construct immobilized thereon.  
     
     
         28 . The apparatus of  claim 27  wherein said temperature-controlled template comprises one or more metal oxide pads affixed to a substrate.  
     
     
         29 . The apparatus of  claim 28 , wherein said metal oxide pads further comprise indium tin oxide (ITO).  
     
     
         30 . The apparatus of  claim 28  wherein said substrate is glass.  
     
     
         31 . The apparatus of  claim 28  wherein said metal oxide pads contain avidin-coated beads.  
     
     
         32 . The apparatus of claim  28 -wherein said metal oxide pads comprise about 10 um by about 10 um to about 100 um by about 100 um.  
     
     
         33 . The apparatus of  claim 28  wherein said metal oxide pads have a resistance of about 50 Ohms.  
     
     
         34 . The apparatus of  claim 28  wherein said template is heated by means of electrodes attached to a metal oxide pad.  
     
     
         35 . The apparatus of  claim 34  wherein said electrodes conduct an applied dc current.  
     
     
         36 . The apparatus of  claim 35  wherein said dc current applied is about 1 mA to about 40 mA.  
     
     
         37 . The apparatus of  claim 28  wherein said metal oxide-substrate is mounted on a water-cooled brass sample plate.  
     
     
         38 . The apparatus of  claim 27  further comprising a means for detecting protein expression.  
     
     
         39 . The apparatus of  claim 38  wherein said means for detecting protein expression is an optical lens.  
     
     
         40 . An in vitro programmable protein micro array comprising a plurality of temperature-controlled metal oxide pads mounted on a substrate wherein each pad is individually temperature-controlled.  
     
     
         41 . The in vitro programmable protein micro array of  claim 40  wherein said temperature-controlled metal oxide pads comprise at least one immobilized nucleic acid construct.  
     
     
         42 . The in vitro programmable protein micro array of  claim 41  wherein said immobilized nucleic acid construct is uniform.  
     
     
         43 . The in vitro programmable-protein micro array of  claim 41  further comprising expressed protein.  
     
     
         44 . The in vitro programmable protein micro array of  claim 43  wherein said expressed protein is localized with said immobilized nucleic acid construct.  
     
     
         45 . The in vitro programmable protein micro array of  claim 41  wherein said temperature-controlled metal oxide pads comprise indium tin oxide pads affixed to a substrate.  
     
     
         46 . The in vitro programmable protein micro array of  claim 45  wherein said substrate comprises glass.  
     
     
         47 . An in vitro programmable protein network comprising a plurality of temperature-controlled metal oxide pads mounted on a substrate wherein each pad is individually temperature-controlled.  
     
     
         48 . The in vitro programmable protein network of  claim 47  wherein said temperature-controlled metal oxide pads comprise at least one immobilized nucleic acid construct.  
     
     
         49 . The in vitro programmable protein network of  claim 48  wherein said immobilized nucleic acid construct is non-uniform.  
     
     
         50 . The in vitro programmable protein network of  claim 47  further comprising expressed protein.  
     
     
         51 . The in vitro programmable protein network of  claim 50  wherein said expressed protein is released from said immobilized nucleic acid construct.  
     
     
         52 . The in vitro programmable protein network of  claim 47  wherein said temperature-controlled metal oxide pads comprise indium tin oxide pads affixed to a substrate.  
     
     
         53 . The in vitro programmable protein network of  claim 52  wherein said substrate comprises glass.  
     
     
         54 . An in vitro method of programming sequential protein expression comprising immobilizing a first nucleic acid construct on a first temperature-controlled template, immobilizing a second nucleic acid construct on a second temperature-controlled template, applying a cell extract and expressing said first protein.  
     
     
         55 . The in vitro method of programming sequential protein expression of  claim 54  wherein said first protein is released from said immobilized nucleic acid construct.  
     
     
         56 . The in vitro method of programming sequential protein expression of  claim 55  wherein said released protein migrates to said second temperature-controlled template.  
     
     
         57 . The in vitro method of programming sequential protein expression of  claim 56  wherein said released protein binds to a protein localized with said second immobilized nucleic acid construct on said second temperature-controlled template.