20.109: 2-component signaling

Module for F'11

 * Remade reporter strain NB462 and tested for induction--using antibiotics at: Amp25, Cam34, Kan10, and including Kan10 always.
 * Remade library K+P- and K-P+. Used H557A as control for K-P+ screen
 * Retested indicator media. Phenol red failed, but Z4 gave clear colonies in dark and red colonies in light (except where dense).

Students will try to identify K-P+ mutants that appear more red in light, i.e. lower units in light, then check to see if dynamic range improved or if all activity diminished

 * Day 1: set up dark/light system in plates, liq culture
 * Day 2: b-gal assay of cultures, set up photo
 * Day 3: transform library and screen, recapitulate setup electronically
 * Day 4: Journal club I, re-streak candidate, 
 * Day 5: DNA for seq, and b-gal assay
 * Day 6: Protein gel and blot, seq data, photo?, other assays
 * Day 7: Probe blot, other assays
 * Day 8: Journal club, data back

Oligos ordered for F'11 screen
5'- CAT ATG GCG GCT GGT GTT AAG CAA CTG GCG GAT GAC CGC ACG CTG CTG ATG RNS GGG GTA AGT CAC GAC TTG CGC ACG CCG CTG ACG CGT -3' 5'- T GAC CGC ACG CTG CTG ATG GCG GGG GTA AGT GCC GAC TTG CGC ACG CCG CTG ACG CGT -3' 5'- TCG TCA ACC TCA TTT TGC GCC AG -3'
 * NO294: K-P+ library, mutagenesis of A553:
 * NO295: K-P+ control, mutagenesis of H557A (CAC to GCC)
 * NO296: fwd seq primer, starts at ~3500 in pCph8
 * 23 mer, Tm = 60.3 ºC

Useful links

 * Original module
 * b-gal assay
 * SDM kitSDM lab protocol
 * controlled swimmingchemotaxis lab protocol
 * APE
 * phenotype arrays
 * q-PCR
 * genetic code
 * Genewiz

K-P+ and K+P- mutations
06.16.09 email from Mike: As for the EnvZ mutants, here's the list we've compiled from the literature. Keep in mind that K+P- really means a shift in the balance of kinase and phosphatase activities and similarly for the K-P+ alleles. None of them is perfectly "clean" in eliminating one of the activities:

K+P-

 * V241G = V555G in Cph8
 * G240E = G554E in Cph8
 * S242D = S556D in Cph8
 * P248Q
 * T247R
 * Q283P
 * Y287D
 * L288P

K-P+
NK note: relevant reference:
 * A239T = A553 in Cph8
 * N343K
 * F390L
 * H243A = H557A in Cph8
 * Mutations that alter the kinase and phosphatase activities of the two-component sensor EnvZ. Hsing W, Russo FD, Bernd KK, Silhavy TJ. J Bacteriol. 1998 Sep;180(17):4538-46. 9721293
 * The role of the G2 box, a conserved motif in the histidine kinase superfamily, in modulating the function of EnvZ. Zhu Y, Inouye M. Mol Microbiol. 2002 Aug;45(3):653-63. 12139613

Primers
NO281 = KP randomized template NO284 = P+ randomized template NO285 = K+ randomized template NO282 = KPLibrary_fwd_1535_1573 NO283 = KPLibrary_rev_1643_1609 NO290 = LibraryBuilder_rev NO279 = GFPfiller_Nde_fwd 5' CATTAGCATATGGGATCCTAAGAGGGCGAGGGCGATGCCACC NO280 = GFPfiller_Mlu_rev 5' CATTAGTGCGCAGATATCTTACTTGTACAGCTCGTCCATGC corrected: 5' CATTAGACGCGTGATATCTTACTTGTACAGCTCGTCCATGC NO288= GFPfiller_Nde_rev  5'CATTAGCATATGGATATCTTACTTGTACAGCTCGTCCATGC NO293= sequencing pCph8 around K+ randomized sequences 5'CTC ATA GCC ACT TTC GGC AGC AAT
 * 5' CATATGGCGGCTGGTGTTAAGCAACTGGCGGATGACCGCACGCTGCTGATG RNS RNS RNS RNS SNW GACTTGCGCACGCCGCTGACGCGT
 * 5' CATATGGCGGCTGGTGTTAAGCAACTGGCGGATGACCGCACGCTGCTGATG RNS GGG GTA AGT SNW GACTTGCGCACGCCGCTGACGCGT
 * 5' CATATGGCGGCTGGTGTTAAGCAACTGGCGGATGACCGCACGCTGCTGATG GCG RNS RNS RNS CAC GACTTGCGCACGCCGCTGACGCGT
 * 5' CAGAAGAATTGCATATGGCGGCTGGTGTTAAGCAACTGG
 * 39 mer
 * Tm (all) = 66.3 ºC
 * Tm (landing, 28 mer) = 63.3 ºC
 * 5' AGGCGAATACGCGTCAGCGGCGTGCGCAAGT
 * 31 mer
 * Tm (all) = 72.1 ºC
 * Tm (landing, 23 mer) = 70.5 ºC
 * 33mer
 * 5' ATC CGC CAG TTG CTT AAC ACC AGC CGC CAT ATG
 * Tm = 67°
 * should hybridize to 5' end of K+ and P+ template oligos (NO284, NO285) to go 'round the horn for library
 * 42 mer
 * Tm (all) = 69.7 ºC
 * Tm (landing, 24 mer) = 67 ºC
 * 41 mer
 * Tm (all) = 64.6 ºC
 * Tm (landing, 23 mer) = 57 ºC
 * Tm (all) = 62.6 ºC
 * Tm (landing, 23 mer) = 57 ºC
 * 21 mer
 * Tm = 60°

Possible ways to epitope tag
All tags at C-terminus, with intention to clone product using unique MfeI and XbaI in pCph8

HA
caattgTGCAGCGTATCGTGGATAACCATAACGGGATGCTGGAGCTTGGCACCAGCGAGCGGGGCGGGCTTTCCATTCGCGCCTGGCTGCCAGTGCCGGTAACGCGGGCGCAGGGCATGACAAAAGAAGGGTACCCGTACGACGTTCCGGACTACGCTTAA'''tctaga
 * 9-amino acid sequence (YPYDVPDYA) recognized by the anti-12CA5 (=anti-HA)
 * reverse translate at |Gene Design with E. coli codon bias
 * default settings gives sequence: TAC CCG TAC GAC GTT CCG GAC TAC GCT

to PCR from DNA2.0 vector

 * NO286=HAfrag_fwd
 * 5' GTCGCTGAACAATTGTGCAGCGTATCGTGG
 * Tm = 61.0 ºC
 * NO287=HAfrag_rev
 * 5' GAACTCGATTGACGTCTAGATTAAGCGTAG
 * Tm = 58.1 ºC

to check insert of HA in pCph8

 * NO289 = Cph8+HAseqprimer_bp135_rev
 * 5' ATTACCGCCTTTGAGTGAGC

His6
caattgTGCAGCGTATCGTGGATAACCATAACGGGATGCTGGAGCTTGGCACCAGCGAGCGGGGCGGGCTTTCCATTCGCGCCTGGCTGCCAGTGCCGGTAACGCGGGCGCAGGGCATGACAAAAGAAGGGcatcaccaccaccatcacTAAtctaga
 * 6 Histidine sequence from pET-45b(+) bp 4963-4980 (Novagen product)