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| ===Week 2===
| | [[Shlo/notebook/summer07 | Summer 2007: iGEM work]]<br> |
| <br>
| | [[Shlo/notebook/fall07 | Fall 2007: iGEM-related work]] |
| 6/27<br>
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| Yesterday night I left some vectors & plasmids to ligate in the 37C incubator. Kevin took the vectors for nucleotide removal, and I speed Vac'd the plasmids. Originally I was going to make a gel and run the plasmids on multiple lanes for gel extraction, but given the large Nanodrop values (242 - 371 ng/uL) and the large amount of Midiprep used (50uL), Bill suggested that I PCR purify them instead, which gives 95% recovery rather than 80% recovery. A gel will still need to be run to confirm that the samples were indeed ligated.<br>
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| *Reconstitution into 30uL of H2O per vector (two vectors total - OmpA1, OmpA2),
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| *split into two samples each (so 15uL total volume)
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| *PCR purified
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| *Eluted with 50uL nuclease-free H2O per tube
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| *prepared a gel using TBE
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| *prepared 5 lanes<br>
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| #Lane 1: 10uL ladder (1kB +)
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| #Lane 2: 1uL OmpA1 plasmid (diluted to 20uL total using H2O)
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| #Lane 3: 3uL OmpA1 plasmid (diluted to 20)
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| #Lane 4: 1uL OmpA2 plasmid (diluted to 20)
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| #Lane 5: 3uL OmpA2 plasmid (diluted to 20)
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| <br> The gel was run at 130V for 45 minutes.<br>
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| 4 tubes total should remain: 2 tubes of each type of plasmid. 2 tubes should have 49uL left (labeled "1"), 2 tubes should have 47uL left (labeled "2")
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| ===Week 1===
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| Doing some reading on Fec now. Really brief notes and some of the more interesting readings follow.<br>
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| Fec genes encode proteins essential for ferric citrate transport in e.coliK12. <br>
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| FecA is an outer membrane protein<br>
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| =N-proximal 79-residue extension: deletion of this extension abolishes induction by ferric citrate but retains feric citrate transport: Kim et al, Transcription induction of the ferric citrate transport genes via the N-terminus of the FecA outer membrane protein, the Ton system, and the electrochemical potential of the cytoplasmic membrane [http://www.blackwell-synergy.com.ezp1.harvard.edu/doi/pdf/10.1046/j.1365-2958.1997.2401593.x?cookieSet=1 Kim article]
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| <br>
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| [http://www.ncbi.nlm.nih.gov.ezp1.harvard.edu/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16718597&ordinalpos=10&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Gene regulation by transmembrane signaling] Some really nice info on structure of Fec and interactions with ferric citrate | |
| <br>
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| [http://biocyc.org/ECOLI/NEW-IMAGE?type=ENZYME&object=EG10292-MONOMER Biocyc]<br>
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| <biblio>
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| #reading1 pmid=11137298<br>
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| #reading2 pmid=12000971
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| </biblio>
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| <br>
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| NanoDrop results (6/20) performed by Ellenor and Stephanie:<br>
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| (1.5 uL used out of a 30uL elution with nuclease free water)<br>
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| S: 10.9 ng/uL<br>
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| S2: 15.4 ng/uL<br>
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| B: 59.1 ng/uL<br>
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| B1: 25.1 ng/uL<br>
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| <br><br><br>
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| Brainstorming for the two-component systems (really for my own use for now - not expected to be coherent)<br>
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| [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10653699&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus Structural comparison of the PhoB and OmpR DNA binding/transactivation domains and the arrangement of PhoB molecules on the phosphate box]<br>
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| -NMR used to determine 3DE structure of PhoB DNA-binding/transactivation domain. Very similar to OmpR DNA-binding/transactivation domain, except for conformation of the long turn region of PhoB (interaction site for sigma subunit, rather than interaction with alpha subunit for OmpR)
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| <br><br>
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| [http://jb.asm.org.ezp1.harvard.edu/cgi/content/full/185/1/317?view=long&pmid=12486069 Interdomain linkers of homologous response regulators determine their mechanism of action]<br> | |
| Focuses on OmpR and PhoB and, as the title suggests, supports that phosphorylation of sites (particularly N-terminus of both proteins) improves affinity to bind DNA. Isolated C terminus of OmpR is insufficient to productively interact with RNA polymerase. <br>
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| I've been told by some of the lab members that OmpR is an inner-membrane protein and therefore cannot be used for our assays. It seems that we'll have to find another protein ...<br>
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| <br>
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| [http://www.ncbi.nlm.nih.gov.ezp1.harvard.edu/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=11325944&ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum The phosphoryl transfer domain of UhpB interacts with the response regulator UhpA] | |
| <br> UhpB = histidine kinase protein that controls production of sugar phosphate transporter UhpT<br>
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| UhpA = response regulator; when phosphate is transferred from histidine to aspartate, ability of kinase to bind to target DNA sequences and to alter gene transcription is altered.
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| <br> Major result: indication that phosphoryl, transfer-dimerization of UhpB participates in specific binding of UhpA, in control of autokinase activity, and dephosphorylation of P-UhpA<br>
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| ... So I found this paper on a search on PubMed for e coli outer membrane protein signaling dimerization. However, I've found that UhpA resides in the cytoplasm, whereas UhpB is an inner-membrane protein. Boo.<br>
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| More thoughts: we could potentially try to target some of these inner-membrane proteins to the outer-membrane. I don't know if this is really feasible - while we can attach the appropriate signal sequence, I'm not sure the environment would allow for correct conformation and activity of said proteins.
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