Edinburgh/Project/Non-antibiotic-Markers/Methods-and-Bibliography

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Methods <a class="cursor-pointer" onclick="expand('all-methods')">(expand)</a>
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Inserting gene into a biobrick vecor: Cloning a PCR product into a biobrick vector protocol on OpenWetWare (http://openwetware.org/wiki/Cfrench:bbcloning) however NEB buffers were used.
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DNA gel preparation: Analysing DNA by gel electrophoresis protocol on OpanWetWare (http://openwetware.org/wiki/Cfrench:AGE) however 0.5*TAE rather than 1*TAE was used.
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Colony PCR screen: Screening colonies by PCR protocol on OpenWetWare http://openwetware.org/wiki/Cfrench:PCRScreening
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Transformations: Preparing and using compenent E.coli cells protocol on OpenWetWare (http://openwetware.org/wiki/Cfrench:compcellprep1)
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PCR reactions : Cloning parts by PCR with Kod polymerase protocol on OpenWetWare (http://openwetware.org/wiki/Cfrench:KodPCR)
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Minipreps : Plasmid DNA minipreps from Escerichia coli JM109 and similar strains protocol on OpenWetWare (http://openwetware.org/wiki/Cfrench:minipreps1)
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Digests to linearise the DNA frangment/determine size of insert: Analytical restriction digests protocol on OpenWetWare (http://openwetware.org/wiki/Cfrench:restriction1)
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DNA purification: Purifying a PCR product from solution protocol on OpenWetWare (http://openwetware.org/wiki/Cfrench:DNAPurification1) however 165 ul NaI, 5 ul glass beads,180 ul wash buffer and 10 ul EB were used.
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DNA preparation for sequencing: 2.5 ul miniprepped DNA, 2 ul water and 1 ul forward primer ( specific for biobrick prefix) or reverse primer (specific for biobrick suffix)
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Nitroreductase activity assay: Overnight liquid cultures of nitroreductase strains were centrifuged at 10000 rpm for 5 mins to pellet the cells. The cells were then resuspended in 250 ul PBS and 1 ul DTT to ensure that cellular proteins are not oxidized. The solution was sonicated 6* (10 s sonication+20 s rest). The supernatant was separated from the pellet by centrifugation and used for the NADH-dependent nitroreductase activity assay.
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To assess background activity NADH (5 ul) and bacterial supernatant (5 ul) were added to 0.8 ml PBS and mixed. OD340 was measured for 1 minute. DNBA(5 ul) was added to the same cuvette to start the reaction and change in OD340 was monitored for 1 minute. DMSO(5 ul) was used a control (DNBA is dissolved in DMSO)
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The protein concentration of each of the supernants was estimated by by Bradford protein assay using the Pierce reagent protocol on OpenWetWare(http://openwetware.org/wiki/Cfrench:ProteinAssay)
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<a class="cursor-pointer" onclick="collapse('all-methods')">Close methods.</a>
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<a name="bibliography">Works Cited</a> <a class="cursor-pointer" onclick="expand('works-cited')">(expand)</a>
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<b>French, C., & Kowal, M.</b> (2010, 09 24). <i>B. subtilis levansucrase. Lethal to E.coli in presence of sucrose.</i> Retrieved 2012, from Registry of standard biological parts: http://partsregistry.org/Part:BBa_K322921
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<b>Gay, P., Coq, D. l., Strinmetz, M., Ferrari, E., & Hoch, J. A.</b> (1983). <i>Cloning Structural Gene SacB, which Codes for Exoenzyme Levansucrase of Bacillus subtilis: Expression of the Gene in Esherichia coli.</i> Journal of Bacteriology , 1424-1431.
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<b>Jahreis, K., Bentler, L., Bockmann, J., Hans, S., Meyer, A., Siepelmeyer, J., et al.</b> (2002). <i>Adaptation of sucrose metabolism in the Escherichia coli Wild-Type Strain EC31132.</i> Journal of Bacteriology, 5307-5316.
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<b>Keuning, S., Janssen, D. B., & Witholt, B.</b> (1985). <i>Purification and Characterisation of Hyrdrolytic Haloalkane Dehalogenase from Xanthobacter autotrophicus GJ10.</i> Journal of Bacteriology, 635-639.
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<b>Naested, H., Fennema, M., Hao, L., Andersen, M., Janssen, D. B., & Mundy, J.</b> (1999). <i>A bacterial haloalkane dehalogenase gene as a negative selectable marker in Arabidopsis.</i> The Plant Journal, 571-576.
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<b>Nicklin, C. E., & Bruce, N. C.</b> (1998). <i>Aerobic degradation of 2,4,6-Trinitrotoluene by Enterobacter cloaceae PB2 and by Pentaerythritol tetranitrate reductase.</i> Applied and environmental microbiology , 2864-2868.
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<b>Nillius, D., Muller, J., & Muller, N.</b> (2011). <i>Nitroreductase (GlNR1) increases susceptibility of Giardia lamblia and Escherichia coli to nitro drugs.</i> Journal of antimicrobial chemotherapy, 1029-1035.
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<b>Kang et al.</b> (2009). <i>"Levan: Applications and Perspectives".</i> Microbial Production of Biopolymers and Polymer Precursors. Caister Academic Press
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<b>Dahech, I, Belghith, K. S., Hamden, K., Feki, A., Belghith, H. and Mejdoub, H.</b> (2011) <i>Antidiabetic activity of levan polysaccharide in alloxan-induced diabetic rats.</i> International Journal of Biological Macromolecules <b>49</b>(4):742-746
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