Team:SDU-Denmark/protocols

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How to amplify DNA from bacteria colonies and solutions <br><br>
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How to amplify DNA from bacterial colonies and solutions <br><br>
''Important remarks'' <br><br>
''Important remarks'' <br><br>
All solutions should be kept at ice until run of PCR <br><br>
All solutions should be kept at ice until run of PCR <br><br>
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=== EX1.1 ===
=== EX1.1 ===
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1. Incubate E. Coli in 110 ml LB media with appropriate antibiotics at 37 oC for 20 hours<br>  
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1. Incubate ''E. Coli'' in 110 ml LB media with appropriate antibiotics at 37 degrees celcius for 20 hours<br> <br>
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2. Harvest cells using centrifugation at 4000 G for 15 min<br>
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2. Harvest cells by centrifugation at 4000g for 15 min<br><br>
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3. Re-suspend cells in 8 mL acetone and sonicate the sample for 2x 30 sek<br>  
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3. Resuspend cells in 8 mL acetone and sonicate the sample for 2x 30 sek<br> <br>
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4. Centrifuge the samples at 16000 G for 2 min and collect 2 mL of the supernatant<br>
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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>
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5. Measure absorbance using UV-Vis spectrophotometer at 450 nm  
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<br><br>
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=== EX1.2 ===
=== EX1.2 ===
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1. Incubate E. Coli in 110 ml LB media with appropriate antibiotics at 37 oC for 20 hours<br>  
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1. Incubate ''E. Coli'' in 110 ml LB media with appropriate antibiotics at 37 degrees celcius for 20 hours<br> <br>
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2. Harvest cells using centrifugation at 4000 G for 15 min<br>
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2. Harvest cells by centrifugation at 4000g for 15 min<br><br>
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3. Re-suspend cells in 8 mL acetone and sonicate the sample for 2x 30 sek<br>  
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3. Re-suspend cells in 8 mL acetone and sonicate the sample for 2x 30 sek<br> <br>
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4. Centrifuge the samples at 16000 G for 2 min and collect 2 mL of the supernatant<br>
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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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• Diluted LB media<br>
• Diluted LB media<br>
• Difco Agar<br>
• Difco Agar<br>
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1mM retinal<br><br>
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1µM retinal (final concentration)<br><br>
''Swimming motility plates''<br>
''Swimming motility plates''<br>
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1. LB media is mixed with 0.3% difco agar and is autoclavated<br>
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1. LB media is mixed with 0.3% difco agar and autoclavated<br><br>
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2. The appropriate antibiotic and 1uM retinal is added to the autoclaved media (NB: for the media used for the control plates, no antibiotic or retinal is added)<br>
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2. The appropriate antibiotic and 1µM retinal (final concentration) is added to the autoclaved media (NB: for the media used for the control plates, no antibiotic or retinal is added)<br><br>
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3. Plates are cast and incubated ON at room temperature<br>
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3. Plates are cast and incubated overnight at room temperature<br><br>
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4. 15 minutes prior to the experiment the plates are incubated at 37°C.<br><br>
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4. 15 minutes prior to the experiment the plates are dried at 37°C.<br><br>
''Sample preparation''<br>
''Sample preparation''<br>
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1. Colony is inoculated in 5mL LB media containing the appropriate antibiotic. The culture is grown ON at 37°C and 180rpm.<br>
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1. 5mL LB media containing the appropriate antibiotic is inoculated with a colony. The culture is grown overnight at 37°C and 180rpm.<br>
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2. ON culture is diluted in 5mL LB media containing the appropriate antibiotic to reach an OD550 of 0.02 and are incubated at 37°C and 180rpm until it reaches an OD550 of 0.5.<br>  
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2. The overnight culture is diluted in 5mL LB media containing the appropriate antibiotic to reach an OD550 of 0.02 and are incubated at 37°C and 180rpm until it reaches an OD550 of 0.5.<br>  
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3. 1mM retinal is added to the culture (NB: no retinal is added to the cultures containing the control cells)<br>
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3. 1µM retinal (final cnocentration) is added to the culture (NB: no retinal is added to the cultures containing the control cells)<br>
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4. The tubes containing the cultures are wrapped in tin foil and are subsequently grown for 2 hours at 37°C and 180rpm.<br><br>
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4. The tubes containing the cultures are wrapped in tin foil (creates darkness) and are subsequently grown for 2 hours at 37°C and 180rpm.<br><br>
''Motility assay''<br>  
''Motility assay''<br>  
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1. 2x2.5uL of culture is placed on each plate, and the plates are placed in a specially engineered lightbox, so that ½ of each plate is illuminated with blue light and the other ½ is kept in dark. 1mL of each culture is used for microscopy.<br>
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1. 2x2.5µL of culture is placed on each plate, and the plates are placed in a specially engineered lightbox, so that ½ of each plate is illuminated with blue light and the other ½ is kept in dark. <br>
2. The plates are incubated at 37°C for 24 hours.<br>
2. The plates are incubated at 37°C for 24 hours.<br>
3. Pictures are taken<br><br>
3. Pictures are taken<br><br>
''Microscopy''<br>
''Microscopy''<br>
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1. 5uL 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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==Growth rate assay==
==Growth rate assay==
===GA1.1===
===GA1.1===
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''Protocol''<br>
''Protocol''<br>
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1. A colony is inoculated in 5mL LB media containing the appropriate antibiotic.<br>
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1. 5mL LB media containing the appropriate antibiotic is inoculated with a colony .<br>
2. The culture is incubated over night at 37°C and 180rpm.<br>
2. The culture is incubated over night at 37°C and 180rpm.<br>
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3. The optical density at 550nm (OD550) of the ON culture is measured and the culture is diluted in 25uL fresh LB media containing the appropriate antibiotic to reach an OD550 of 0.02. The culture is incubated for 24 hours at 37°C and 180rpm.<br>
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3. The optical density at 550nm (OD550) of the overnight culture is measured and the culture is diluted in 15mL fresh LB media containing the appropriate antibiotic to reach an OD550 of 0.02. The culture is then incubated at 37°C and 180rpm.<br>
4. OD550 of the colony is measured every hour for the first 12 hours, and after 24 hours.<br><br>  
4. OD550 of the colony is measured every hour for the first 12 hours, and after 24 hours.<br><br>  
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==Flagella staining==
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===1.1===  
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== Flagella staining ==
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<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>
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===FS1.1===  
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Day 1: The bacteria were grown in 5 ml-LB media ON. The solutions used for staining were prepared.
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Day 1: The bacteria were grown in 5 ml-LB media overnight. The solutions used for staining were prepared.
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The following components were added in the listed order:<br>
The following components were added in the listed order:<br>
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• 5 g of tannic acid was dissolved in 9.65 ml distilled water.<br>
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• 5 g of tannic acid dissolved in 9.65 ml distilled water.<br>
• 150 µl  9% FeCl3<br>
• 150 µl  9% FeCl3<br>
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• 10% aqueous ammonia solution was added until the silver nitrate was dissolved. Approximately 2 ml.  
• 10% aqueous ammonia solution was added until the silver nitrate was dissolved. Approximately 2 ml.  
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Day 2: The bacteria were boosted in 5 ml LB-media to ensure that they were in the exponential growth phase when used for staining. They were diluted to approximately OD550 1.  
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Day 2: The bacteria were boosted in 5 ml LB-media to ensure that they were in the exponential growth phase when used for staining. They were diluted to approximately an OD550 of 1.  
<br><br>
<br><br>
Preliminary bacteria work:<br>
Preliminary bacteria work:<br>
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===1.2===
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===FS1.2===
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Day 1: Bacteria were platede on agar plates and incubated at 37 degrees ON.  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 2: A bacteria colony was dissolved in LB-media.  
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Day 2: A bacterial colony was dissolved in LB-media.  
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Materials
Materials
<br><br>
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*         LB media
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        LB media <br>
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*         LA plates
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        LA plates <br>
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*         LA plates with appropriate antibiotic
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        LA plates with appropriate antibiotic<br>
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*         0.9% NaCl
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        0.9% NaCl<br>
<br>
<br>
   
   
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Protocol
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'''Protocol'''
<br><br>
<br><br>
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1.      A bacteria colony is inoculated in 5mL LB media with appropriate antibiotic.
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1.      5mL LB media with appropriate antibiotic is inoculated with a bacterial colony.
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2.      Culture is incubated over night at 30°C and 180rpm
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2.      The culture is incubated over night at 30°C and 180rpm
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3.      100uL of the culture is serial diluted in 900uL 0.9% NaCl to reach a dilution of 107.
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3.      100µL of the culture is serially diluted in 900µL 0.9% NaCl until a dilution of 10<sup>7</sup> is reached.
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4.      100uL of the 105, 106 and 107 dilution is spreaded on LA plates, and LA plates with the appropriate antibiotic, respectively.
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4.      100µL of the 10<sup>5</sup>, 10<sup>6</sup> and 10<sup>7</sup> dilutions are spread onto LA plates, and LA plates with the appropriate antibiotic, respectively.
<br>
<br>
5.      Plates are incubated at 37°C for 16-24 hours. The following day the colonies formed on the plates are counted and cfu are determined.
5.      Plates are incubated at 37°C for 16-24 hours. The following day the colonies formed on the plates are counted and cfu are determined.
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6.      500uL of the 105 dilution is added to 4.5mL of fresh LB media without any antibiotics.
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6.      500µL of the 10<sup>5</sup> dilution is added to 4.5mL of fresh LB media without any antibiotics.
<br>
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7.      The new culture is incubated over night at 30°C and 180rpm
7.      The new culture is incubated over night at 30°C and 180rpm
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8.      The experiment is carried out for 5 days. (NB: antibiotic is only added to the first culture. The remaining days the bacteria are grown in cultures without any antibiotics)
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8.      The experiment is carried out for 5 days.  
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<br>(NB: antibiotic is only added to the first culture. The remaining days the bacteria are grown in cultures without any antibiotics)
<br><br>
<br><br>
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==Scanning Electron Microscope ==
==Scanning Electron Microscope ==
===SEM 1.1===
===SEM 1.1===
<br>
<br>
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Day 1: The bacteria was cultivated ON in 5 ml LB-media at 37 degrees. <br><br>  
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Day 1: The bacteria was cultivated overnight in 5 ml LB-media at 37 degrees celcius. <br><br>  
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Day 2: The ON culture was centrifuged  15 min at 4000prm. Afterwards the pellet was resuspended in  distilled water. We aimed to get approximately 106 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 appeared when sample were exposed to the vacuum in the electron microscope.  <br><br>
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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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__TOC__
__TOC__
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<div id="bonus">
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<br>
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<br>
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<br>
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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