IGEM:Cambridge/2008/Notebook/Voltage/Gene Design

=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,C,D,E Where F-C are the functional membrane protein subunits, and D-E comprises a bacterial 2-component regulatory system.




 * 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.
 * The D-E 2-component system consists of a membrane protein turgidity sensor and a transcription factor. It controls Kdp operon expression in vivo, by reducing gene expression when turgor is high.
 * Since we wish to over-express Kdp, we decided not to include the regulatory system in our biobrick. (Osmotic buffering would be used instead.)

Amplification from E.coli MG1655
-Forward: ATAT GAATTC ATAT TCTAGA TGAGTGCAGGCGTGATAACCGGCGTATT EcoRI       XbaI
 * This was performed via PCR amplification of the genome template using the following primers:

-Reverse: CTCT CTGCAG CTCT ACTAGT TTATTCATCAAGTTTATCCAGCGCCAGAT PstI        SpeI
 * Primer overhangs incorporated the biobrick prefix and suffix into the section, restriction sites shown in bold.
 * The result of this PCR is shown below:

Integration into Vector

 * The vector used was low copy-number plasmid pSB4C5, with chloramphenicol resistance and a death gene as selection markers.
 * Kdp PCR product and pSB4C5 were both cut with EcoRI & SpeI, (vector backbone was dephosphorylated to prevent circularisation) then ligation into the vector can occur as shown.




 * 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.

=Promoter+RBS Biobrick=

Promoter

 * The promoter chosen for use with Kdp was OsmY (Part BBa_J45992).
 * It is a stationary phase promoter, and since we require high cell densities in our final "voltage measurement" medium, we want Kdp to only be expressed in stationary phase.
 * This will reduce the metabolic and osmotic stress on dividing cells in exponential phase.

Ribosome Binding Site

 * Three different strength RBSs were investigated, B0030, B0031 and B0032.
 * B0030 is the strongest(15bp length), B0031 medium(14bp) and B0032 weakest(13bp).
 * Investigating three will help us determine the optimum levels of Kdp expression.

Amplification from E.coli MG1655
-Forward: CTAT GAATTC ATAT TCTAGA GCTGGCACAGGAACGTTATCC (All OsmY-RBS constructs) EcoRI       XbaI
 * These parts were extracted using PCR from the Registry of Standard Biological Parts. However, the RBS biobricks are so small that we built their sequences into the reverse primers.
 * The primer sequences used are:

-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


 * PCR with these primers creates three different promoter-RBS biobrick parts (OsmY-B003x):



Integration into Vector

 * The vector used was low copy-number plasmid pSB4C5, with chloramphenicol resistance and a death gene as selection markers.
 * OsmY-B003x and pSB4C5 were both cut with XbaI & SpeI, (vector backbone was dephosphorylated to prevent circularisation) then ligation into the vector can occur as shown:






 * 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=
 * 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-OsmY-B003x with PstI first, then SpeI, in order to make sure Pst cuts correctly.
 * Ligation will form the following functional plasmid:



=GluR0 Biobrick=

Gene Selection

 * In order to create a measurable voltage change when a chemical was "recognised" we decided to use an ionotropic ligand-gated potassium efflux channel that binds glutamate. This also simulates the action of glutamate as a neurotransmitter in the CNS.
 * The gene chosen comes from the cyanobacteria Synechocystis sp. PCC 6803. This species is gram negative (similar to E.coli) and is used as a paradigm for evolutionary research concerning the AMPA receptor proteins.
 * GluR0 is a well characterised glutamate-gated K+ membrane channel, which has a considerable degree of structural and functional homology to rat neurone GluR2 AMPA receptors, see the paper Functional Characterisation of a Glutamate-gated Potassium Channel

DNA Synthesis

 * The protein sequence was obtained via NCBI from the Synechocystis sp. PCC 6803 genome.
 * The gene sequence could not be directly used because of codon optimisation problems (Synechocystis uses many codons that are "rare" in E.coli"
 * The protein sequence was back-translated to DNA using GeneDesigner™ and codons were assigned using the E.coli usage table.
 * The promoter BBa_J23116, RBS BBa_J61117 and Biobrick prefix and suffix sequences were added to the design.
 * Finally, unwanted restriction sites within the gene(EcoRI, XbaI, SpeI and PstI) were manually removed by selecting alternative codons for any given amino acid.
 * The gene was synthesised and sequenced by DNA2.0 into one of their standard vectors.