IGEM:Cambridge/2008/Notebook/Voltage/Gene Design
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(New page: =KdpF-C Biobrick= ==Gene Selection== *Kdp is a well documented P-Type K+ ATPase found naturally in E.coli, used to actively pump ions into the cell. *It consists of a 6-gene operon: F,A,B...) |
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+ | The two constructs that we designed are detailed bellow. KDP is the potassium importer and GluR0 is the glutamate gated potassium channel. Details of their construction can be found here: | ||
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+ | [[IGEM:Cambridge/2008/Notebook/Voltage/BioBrick_Manipulation| KDP]] | ||
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+ | [[IGEM:Cambridge/2008/Notebook/Voltage/GluR0_Manipulation| GluR0]] | ||
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+ | =KdpF-C Biobrick- [http://partsregistry.org/Part:BBa_K090003 BBa_K090003]== | ||
==Gene Selection== | ==Gene Selection== | ||
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*It consists of a 6-gene operon: F,A,B,C,D,E Where F-C are the functional membrane protein subunits, and D-E comprises a bacterial 2-component regulatory system. | *It consists of a 6-gene operon: F,A,B,C,D,E Where F-C are the functional membrane protein subunits, and D-E comprises a bacterial 2-component regulatory system. | ||
- | [[Image:kdpfull.JPG |800px| Native operon context from NCBI]] | + | [[Image:kdpfull.JPG |800px |Native operon context from NCBI]] |
*Literature shows that Kdp acts as a high-affinity transport system, and works most effectively at low external potassium concentrations, where a change in ion flux would be most likely to produce a measurable voltage difference. | *Literature shows that Kdp acts as a high-affinity transport system, and works most effectively at low external potassium concentrations, where a change in ion flux would be most likely to produce a measurable voltage difference. | ||
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*Note: pSB4C5_Kdp biobrick plasmid has no promoter/RBS and so Kdp is not expressed in transformants. | *Note: pSB4C5_Kdp biobrick plasmid has no promoter/RBS and so Kdp is not expressed in transformants. | ||
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=Promoter+RBS Biobrick= | =Promoter+RBS Biobrick= | ||
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*Note: This promoter-RBS construct did not cause unwanted transcript problems because there are many double terminators scattered throughout the pSB4C5 backbone. | *Note: This promoter-RBS construct did not cause unwanted transcript problems because there are many double terminators scattered throughout the pSB4C5 backbone. | ||
- | =Combination of Kdp, OsmY and B003x= | + | =Combination of Kdp, OsmY and B003x to make [http://partsregistry.org/Part:BBa_K090004 BBa_K090004]= |
*This will create a functional biobrick plasmid in which Kdp is overexpressed only in stationary phase of growing cells. | *This will create a functional biobrick plasmid in which Kdp is overexpressed only in stationary phase of growing cells. | ||
:*Cut pSB4C5-Kdp with XbaI and PstI | :*Cut pSB4C5-Kdp with XbaI and PstI | ||
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:*Ligation will form the following functional plasmid: | :*Ligation will form the following functional plasmid: | ||
- | [[Image:psb4c5_osmy_b0030_kdp.JPG|400px]] | + | [[Image:psb4c5_osmy_b0030_kdp.JPG|400px]] |
- | =GluR0 Biobrick= | + | =GluR0 Biobrick- [http://partsregistry.org/Part:BBa_K090002 BBa_K090002]= |
==Gene Selection== | ==Gene Selection== | ||
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[[Image:Glur0plasmid.JPG |800px]] | [[Image:Glur0plasmid.JPG |800px]] | ||
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Latest revision as of 22:30, 29 October 2008
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The two constructs that we designed are detailed bellow. KDP is the potassium importer and GluR0 is the glutamate gated potassium channel. Details of their construction can be found here:
KdpF-C Biobrick- BBa_K090003=Gene Selection
Amplification from E.coli MG1655
-Forward: ATAT GAATTC ATAT TCTAGA TGAGTGCAGGCGTGATAACCGGCGTATT EcoRI XbaI -Reverse: CTCT CTGCAG CTCT ACTAGT TTATTCATCAAGTTTATCCAGCGCCAGAT PstI SpeI
Integration into Vector
Promoter+RBS BiobrickPromoter and RBS SelectionPromoter
Ribosome Binding Site
Amplification from E.coli MG1655
-Forward: CTAT GAATTC ATAT TCTAGA GCTGGCACAGGAACGTTATCC (All OsmY-RBS constructs) EcoRI XbaI -B0030 Reverse: CGCG CTGCAG CTCT ACTAGT (TTTCTCCTCTTTAAT)TTGTTAAATATAGA PstI SpeI B0030 -B0031 Reverse: CTCT CTGCAG CTCT ACTAGT (GGTTTCCTGTGTGA)TTGTTAAATATAGAT PstI SpeI B0031 -B0032 Reverse: CTCT CTGCAG CTCT ACTAGT (CTTTCCTGTGTGA)TTGTTAAATATAGATCA PstI SpeI B0032
Integration into Vector
Combination of Kdp, OsmY and B003x to make BBa_K090004
GluR0 Biobrick- BBa_K090002Gene Selection
DNA Synthesis
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