Team:SDU-Denmark/protocols

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(Flagella staining)
 
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3. Resuspend cells in 8 mL acetone and sonicate the sample for 2x 30 sek<br> <br>
3. Resuspend cells in 8 mL acetone and sonicate the sample for 2x 30 sek<br> <br>
4. Centrifuge the samples at 16000g for 2 min and collect 2 mL of the supernatant<br><br>
4. Centrifuge the samples at 16000g for 2 min and collect 2 mL of the supernatant<br><br>
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5. Measure absorbance using UV-Vis spectrophotometer at 450 nm, was preformed on a …. From MEMPHYS<br><br>
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5. Measure absorbance using UV-Vis spectrophotometer at 450 nm  
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3. Re-suspend cells in 8 mL acetone and sonicate the sample for 2x 30 sek<br> <br>
3. Re-suspend cells in 8 mL acetone and sonicate the sample for 2x 30 sek<br> <br>
4. Centrifuge the samples at 16000g for 2 min and collect 2 mL of the supernatant<br><br>
4. Centrifuge the samples at 16000g for 2 min and collect 2 mL of the supernatant<br><br>
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5. Measure absorbance using an HPLC at 450 nm for bata-carotene and 382 nm for retinal analysis, using a C18… column with 100% Methanol as the A Buffer and 60% Methanol, 40% acetone as the B buffer <br><br>
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5. Measure absorbance using an HPLC at 450 nm for bata-carotene and 382 nm for retinal analysis, For this particular purpose, we use a Poroshell 120 EC-C18 (4,6 x 150 mm 2,7 micron)column, and the eluents used are as follows: <br>
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A-buffer: 100% methanol with 0,1% trifluoroacetic acid. <br>
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B-buffer: A mixture consisting of 60% methanol and 40% acetone with 0,1% trifluoroacetic acid. <br>
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Due to the chemical properties of beta-carotene and retinal, respectively, retinal will come through the column before beta-carotene when a gradient is run from 100% A-buffer to 100% B-buffer. <br>
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Afterwards, the solutions of purified retinal or beta-carotene are studied using UV-vis photospectrometry and the values and spectra are compared to those of the same compounds of known concentrations. Again, this gives both qualitative and quantitative indications of whether the compound in question is present and, if it is, in what concentration. Usage of the HPLC and instruction on how to use it was kindly provided by [http://www.sdu.dk/Om_SDU/Institutter_centre/C_FLinT FLINT]<br>
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''Microscopy''<br>
''Microscopy''<br>
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1. 5µL cell culture is used for the microscopy<br>
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1. 5µL of bacterial culture is placed in the center of a microscopy slide dimensions 7.5cm x 1.5cm and a cover slide is used to cover the culture.<br>
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2. To avoid laminar flow, the microscopy slide is sealed with nail polish.<br>
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2. To eliminate any flow in the system, which can be mistaken for bacterial motility, the cover slide is sealed with ordinary mail polish.<br>
3. Samples are examined under the microscope.<br><br>
3. Samples are examined under the microscope.<br><br>
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=== PS1.2 ===
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A protocol for optimizing the motility of E.coli MG1655 to use for microscopy<br>
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This protocol is designed based on preceeding pilot studies<br>
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''Materials:''<br>
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• LB media<br>
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• Motility buffer (20mM potassium phosphate and 0.1mM EDTA dissolved in ddH2O [[https://2010.igem.org/Team:SDU-Denmark/protocols#References 1]]<br>
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• 1mM retinal<br>
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''Protocol:''
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1. A colony is inoculated in 5mL LB media with appropriate antibiotic.<br>
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2. The culture is incubated for 12h. at 22° and 160rpm<br>
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3. The cultures containing the unmodified bacteria (controls) is then diluted 1:10 in motility buffer, at which point these are ready for microscopy <br>
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4. The culture containing the modified bacteria is added 1uM retinal (final concentration), and is incubated for an additional 2h in darkness at 22°C and 160rpm.<br>
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5. The culture is then diluted 1:10 in motility buffer and are ready for microscopy.<br>
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''Microscopy''<br>
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1. 5µL of bacterial culture is placed in the center of a microscopy slide dimensions 7.5cm x 1.5cm and a cover slide is used to cover the culture.<br>
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2. To eliminate any flow in the system, which can be mistaken for bacterial motility, the cover slide is sealed with ordinary mail polish.<br>
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3. Samples are examined under the microscope.<br><br>
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== Flagella staining ==
== Flagella staining ==
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The following protocols are based on knowledge recived from the article: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC44660 Dimorphic transition in Escherichia coli and Salmonella typhimurium: surface-induced differentiation into hyperflagellate swarmer cells] <br>
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The following protocols are based on knowledge recived from [[https://2010.igem.org/Team:SDU-Denmark/protocols#References 2]]<br>
===FS1.1===  
===FS1.1===  
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===FS1.2===
===FS1.2===
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Day 1: Bacteria were platede on agar plates and incubated at 37 degrees overnight.  The staining solutions were prepared.<br><br>
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Day 1: Bacteria were platede on agar plates and incubated at 37 degrees celcius overnight.  The staining solutions were prepared.<br><br>
Solution I: <br>
Solution I: <br>
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Day 1: The bacteria was cultivated overnight in 5 ml LB-media at 37 degrees celcius. <br><br>  
Day 1: The bacteria was cultivated overnight in 5 ml LB-media at 37 degrees celcius. <br><br>  
Day 2: The overnight culture was centrifuged  15 min at 4000prm. Afterwards the pellet was resuspended in  distilled water. We aimed to get approximately 10<sup>6</sup> bacteria in 10 µl solution which was plated on double adhesive tape at the top of the grid. The solution was allowed to air dry and the remaining fluid disappeared as samples were exposed to the vacuum in the electron microscope.  <br><br>
Day 2: The overnight culture was centrifuged  15 min at 4000prm. Afterwards the pellet was resuspended in  distilled water. We aimed to get approximately 10<sup>6</sup> bacteria in 10 µl solution which was plated on double adhesive tape at the top of the grid. The solution was allowed to air dry and the remaining fluid disappeared as samples were exposed to the vacuum in the electron microscope.  <br><br>
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The sample was examined with different electron intensity and magnification. We found that the best picture was taken with a electron intensity of 10 kv.
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The sample was examined with different electron intensity and magnification. We found that the best picture was taken with a electron intensity of 10 kV  and a magnitude on 10kx.<br><br>
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== Charactarization of K389016 (VirA/G reporter device mRFP) ==
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===CK1.1===
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''Materials:''<br>
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•    20mM acetosyringone (39.3 acetosyringone dissolved in 1mL DMSO and 9mL ddH2O)<br>
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•    LB media<br>
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•    35µg/mL chloramphenicol<br>
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''Protocol:''<br>
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1.    A colony is inoculated in 5mL LB media with 35µg/mL chloramphenicol and incubated over night at 37°C and 180rpm.<br>
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2.    Parallel cultivations of 25mL LB media, 35µg/mL chloramphenicol and acetosyringone concentrations of 0uM (control), 100uM, 200uM and 400uM respectively.<br>
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3.    The cultures were incubated in a waterbath of 37°C and 180rpm<br>
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4.    The optical density at 550nm (OD550) was measured every two hours and samples were freezed at -80°C and used for fluorescence measurements (excitation at 584nm, emmitation at 607nm)<br><br>
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== References ==
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[1]Khan S, Amoyaw K, Spudich JL, Reid GP, Trentham DR,[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1260487/pdf/biophysj00100-0082.pdf Bacterial chemoreceptor signaling probed by flash photorelease of a caged serine], Biophys J. 1992 Apr;62(1)<br>
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[2]Harshey RM, Matsuyama T,[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC44660/pdf/pnas01140-0333.pdf Dimorphic transition in Escherichia coli and Salmonella typhimurium: Surface-induced differentiation into hyperflagellate swarmer cells],Proc Natl Acad Sci U S A. 1994 August 30; 91(18): 8631–8635<br>
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Almost like cake recipes... although cake is infinitely more delicious than bacteria.
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Almost like cake recipes... although cake is infinitely more delicious than bacteria. <br><br>
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Get a template for calculating your ligation concentrations <br><html><a href="https://static.igem.org/mediawiki/2010/4/40/Team_SDUTemplate_for_ligation.ZIP" style="color: #50CC38; text-decoratio: bold; font-size: 24px;">HERE</a></html>
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[https://2010.igem.org/Team:SDU-Denmark/project/activities/commoninterest/diller 8===D]
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Latest revision as of 03:58, 28 October 2010