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| ===Week 2===
| | [[Shlo/notebook/summer07 | Summer 2007: iGEM work]]<br> |
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| | [[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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| The plasmids are Lpp+OmpA1+pet29 and Lpp+OmpA2+pet29.<br>
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| *Reconstitution into 30uL of H2O per plasmid (two plasmids total - OmpA1, OmpA2),
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| *split into two samples each (so 15uL total volume)
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| *PCR purified (PCR purification kit from Qiagen)
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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"). After gel ran for 45 minutes, bands appeared about 3/4 way down the gel. Ethidium bromide (2uL) and 100mL TBE buffer used, put on shaker.
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| [[Image:gelpostpcrplasmid0627.jpg]]
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| Idea: increasing ligation efficiency<br>
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| According to a paper I just read, (Lund et al) "temperature cycle ligations" may provide a 4-8 fold increase on cloning efficiency, since the cycling balances high enzyme activity and DNA annealing. The temperature cycle should run for 12-16 hours, cycling between 30 second bouts at 10C and 30C. <br>
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| <br><b>Readings toward QS and lux</b>
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| [http://departments.kings.edu/biology/lux/bacterial.html Bacterial Transformation Experiment] James Slock has a detailed protocol for transforming the lux genes into e.coli. Actually, the transformation protocol is the same as what we've been doing, but there is some specific info on lux stuff. Not really essential, I guess.<br> | |
| [http://parts.mit.edu/registry/index.php/Featured_Parts:Cell-Cell-Signaling MIT Parts Registry: Overview of LuxR system] | |
| ----
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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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| [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 | |
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| [http://biocyc.org/ECOLI/NEW-IMAGE?type=ENZYME&object=EG10292-MONOMER Biocyc]<br> | |
| <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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| 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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| 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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| [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>
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| 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]
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| <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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