User:Tk/Notebook/MF-xfm/2008/04/05

MF-xfm

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Kenri04: fluorescent ECFP and EYFP proteins in M. pneumoniae Used the M. pneumoniae tuf (elongation factor Tu) promoter with these primers: 789547 ATTTTGCAAACTGATGACAA 789528 789312 TTCTCTCTTGCCATGTGTTTG 789332 FEATURES Location/Qualifiers source 1..236 /organism="Mycoplasma pneumoniae M129" /mol_type="genomic DNA" /strain="M129; ATCC 29342" /db_xref="ATCC:29342" /db_xref="taxon:272634" gene complement(1..>138) /locus_tag="MPN_666" /note="synonym: K05_orf251" CDS complement(1..>138) /locus_tag="MPN_666" /note="MPN666(new), 176(Himmelreich et al., 1996)" /codon_start=1 /transl_table=4 /product="conserved hypothetical protein" /protein_id="AAB95824.1" /db_xref="GI:1673838" /translation="MEQNNCKIRTWFAKLALAQKVFLGVIPLFFICFVFVIADIVISL QNKGHIIEEIDKFTNQSNVMLLIYACWYVSKPKSHYLKNQQFFLSAFAYIIFTFLGYN VILAASQQAYSDKDAYSLASSVFLHVLAPIAFLVAGIVKMKTDKDVTFNHFWKSLGYF MIYPLVYGLYLATIPYVRGHYVSDDGKSTTYVVYGEITNTKDNPIVAWPVVICFLFIY FPLSFLAVYALQCKLLNRPLKAQFKCATNKCPK" gene <223..236 /gene="tuf" /locus_tag="MPN_665" /note="synonym: K05_orf394" CDS <223..236 /gene="tuf" /locus_tag="MPN_665" /note="MPN665(new), 177(Himmelreich et al., 1996)" /codon_start=1 /transl_table=4 /product="elongation factor TU" /protein_id="AAB95825.1" /db_xref="GI:1673840" /translation="MAREKFDRSKPHVNVGTIGHIDHGKTTLTAAICTVLAKEGKSAA TRYDQIDKAPEEKARGITINSAHVEYSSDKRHYAHVDCPGHADYIKNMITGAAQMDGA ILVVSATDSVMPQTREHILLARQVGVPRMVVFLNKCDIATDEEVQELVAEEVRDLLTS YGFDGKNTPIIYGSALKALEGDPKWEAKIHDLMNAVDEWIPTPEREVDKPFLLAIEDT MTITGRGTVVTGRVERGELKVGQEIEIVGLRPIRKAVVTGIEMFKKELDSAMAGDNAG VLLRGVDRKEVERGQVLAKPGSIKPHKKFKAEIYALKKEEGGRHTGFLNGYRPQFYFR TTDVTGSISLPENTEMVLPGDNTSITVELIAPIACEKGSKFSIREGGRTVGAGSVTEV LE" ORIGIN 1 attttgcaaa ctgatgacaa tgtcagcaat tacaaaaaca aaacaaataa aaaataaggg 61 aattaccccc aagaagacct tttgtgctaa cgccagtttg gcaaatcaag ttctgatttt 121 gcaattattt tgctccatat gaattacact actccaagaa ttataagcct ctctacagct 181 ttatctcaaa cttatgtaaa attagagacg taattcaaac acatggcaag agagaa // >gi|26117688:c789547-789312 Mycoplasma pneumoniae M129, complete genome ATTTTGCAAACTGATGACAATGTCAGCAATTACAAAAACAAAACAAATAAAAAATAAGGGAATTACCCCC AAGAAGACCTTTTGTGCTAACGCCAGTTTGGCAAATCAAGTTCTGATTTTGCAATTATTTTGCTCCATAT GAATTACACTACTCCAAGAATTATAAGCCTC TCTACA GCTTTATCTCAAACTTA TGTAAAATT AGAGACG TAATTCAAACAC ATG GCA AGA GAG AAa ttt gac cga tct aaa ccc ca The ATG start annotated for tuf in the genbank entry may not be correct. See the lack of an RBS, while the end sequence shown here is an excellent RBS. The ttg following the RBS would be an excellent start, and presumably Kenri and Miyata also thought that was the real start codon. The promoter shows an excellent -10 and -35, though the spacing is a bit long (19 bp). There is also a TG just 5' of the -10, as is typical for strong mycoplasma promoters. This almost certainly wrong -- the ttg is not in frame; also, the initial aa's of EF-Tu are highly conserved, so unless they are all mis-annotated, this is wrong. The ttt (phenylalanine) could conceivably be the real start. This looks like an interesting paper: Gaucher,E.A., Thomson,J.M., Burgan,M.F. and Benner,S.A. TITLE Inferring the palaeoenvironment of ancient bacteria on the basis of resurrected proteins JOURNAL Nature 425 (6955), 285-288 (2003) PMID 13679914 Transformation results of electroporation tests plated 100 μl of a 1 ml outgrowth volume Zillions of colonies from a 1 ng pUC19 transformation. Colonies visible in 7-8 hours under the scope. transformations from 1/2 hour grown and 2 hour grown under non-selective conditions seemed identical. Transformations with four samples of transposases (made some time ago and stored at -20) 100 ng/μl tpase + 100 ng DNA -- 1/2 hour growth: 5 colonies -- 2 hour growth, 31 200 ng/μl tpase -- 1/2 hour 17; -- 2 hour 63 500 ng/μl tpase -- 1/2 hour 27; -- 2 hour 128 Epicentre tpase -- 1/2 hour 34; -- 2 hour about 200 transformation efficiency: input DNA 25 ng (100 ng in 4 μl transposome volume) Outgrowth portion plated 0.1 with 100 colonies, this is 1000 transformants per 25 ng, or 40000 per microgram of DNA, or 4E4/μg Goryshin00 claims he gets 1E8 transformants in MC1061 vs. 1E9 for plasmid transformation The QC test for Epicentre is 1E5/μg for electrocompetent cells that are 1E10 competent with pUC19