Team:Bielefeld-CeBiTec/Notebook/Miscellaneous
Miscellaneous
- generating of sfGFP and mRFP lysate
- over night culture of e.coli with mRFP Plasmid or rather sfGFP
- inoculation of 100 mL culture in a 1 L flask
- induction with rhamnose to produce mRFP or rather sfGFP, except one culture
- cell harvest after 6 hours
- resuspension of the pellet with 50 mM Hepes pH 8
- sonication with the HD 2070 Sonopuls from Bandelin, 6 times for 2 minutes on ice
- centrifugation for 30 min, 5000 rpm and 4°C.
- take lysate and discard the pellet
- fluorescence imaging
- we took lysate with sfGFP, lysis buffer and purified GFP
- We got a filter from light engineering from the women cultural center Bielefeld e.V. ("Frauenkulturzentrum Bielefeld e.V.") and put it in front of the smartphone.
- the picture showed great evidence for possible fluorescence imaging with the right filters
This Week we started with the iGEM 2015 Measurement Interlab Study:
- Heat shock transformation of BioBricks from the distribution:
- BBa_K823005 (Anderson promoter J23101)
- BBa_K823008 (Anderson promoter J23106)
- BBa_K823013 (Anderson promoter J23117)
- BBa_I13504 (GFP)
- we made a preselection on basis of the light transmitting spectra of the filters, we bought
- we testet DEEP PURPLE BLUE (797), MEDIUM PURPLE (049) MAUVE (126), CHRYSALIS PINK (798), CONGO BLUE (181), ULTIMATE VIOLET (707), SPECIAL KH LAVENDER (799), TOKYO BLUE (071), J WINTER BLUE (713), PALACE BLUE (198), DEEP BLUE (120) and SPECIAL MEDIUM BLUE (363) as possible filters in front of the flash
- As possible filter in front of the camera we tested VELVET GREEN (735), FLUORESCENT GREEN (219), PRIMARY GREEN (139), DARK YELLOW GREEN (090) and TWICKENHAM GREEN (736)
- we filled reactions tubes with lysis buffer, lysate without sfGFP, sfGFP lysate and purified GFP, each tube were filled with 500 µL and we took an empty reaction tube.
- to get pictures under same conditions we took a carton and put black carton in it, to get an dark environment
- we took pictures of the tube with the different filter combinations
- afterwards we analyzed the pictues with the image processing software Fiji
- we draw a square in the pictures and took it to the relevant places where we wanted to measure the fluorescence
- you can get the measurement results from fiji and calculate the fluorescence with the following formula: fluorescence = Integrated Density – (Area of selected X Mean fluorescence of background readings)
- we took the results and analyzed them and made the conclusion that, twickenham green in combination with tokyo blue is the best combination to photograph gfp with your smartphone with less background as possible
- fluorescence imaging of GFP
iGEM 2015 Measurement Interlab Study:
- Colonies from the BioBrick Transformation were used to inoculate over night cultures
- 5 ml LB-Medium + Cm (20 µg/ml), 37°C
- Mini-preps of KRX(BBa_K823005), KRX(BBa_K823008) and KRX(BBa_K823013)
- Plasmids stored at -20°C
- Second heat shock transformation of chem. competent KRX with BBa_I13504
- Colonies were used to inoculate over night cultures (5 ml LB + Cm20, 37°C)
- fluorescence imaging of mRFP
- we made a preselection on basis of the light transmitting spectra of the filters, we bought
- DEEP ORANGE (158), DEEP GOLDEN AMBER (135), TERRY RED (781), MADGE (507), FIRE (019), FLAME RED (164), LIGHT RED (182) and PRIMARY RED (106) as possible filters in front of the flash
- As possible filter in front of the camera we tested PRIMARY GREEN (139), DARK YELLOW GREEN (090), AURORA BOREALIS GREEN (740) and TWICKENHAM GREEN (736)
- we filled reactions tubes with lysis buffer, lysate without mRFP and mRFP lysate each tube were filled with 500 µL and we took an empty reaction tube.
- to get pictures under same conditions we took a carton as in the week before
- we took pictures of the tube with the different filter combinations
- afterwards we analyzed the pictues with the image processing software Fiji
- we draw a square in the pictures and took it to the relevant places where we wanted to measure the fluorescence
- you can get the measurement results from fiji and calculate the fluorescence with the following formula: fluorescence = Integrated Density – (Area of selected X Mean fluorescence of background readings)
- we took the results and analyzed them and made the conclusion that, twickenham green in combination with light red is the best combination to photograph mRFP with your smartphone with less background as possible
iGEM 2015 Measurement Interlab Study:
- Mini-prep of KRX(BBa_I13504)
- Plasmid stored at -20°C
- Fluorescence Imaging
- Fluorescence imaging of fluids is possible. But we want to design a paper based test strip, therefore we had to test if fluorescence imaging on paper works also
- We made different dilutions out of our sfGFP lysat, mRFP lysat, lysat without mRFP/sfGFP and purified GFP
- we took the different papers (Macherey and Nagel MN872B, Munktell C350L, Munktell FN 3 and filter paper from Merck)
- we made pieces of paper with the same size with a hole-punch
- we put 10 µL from the different dilutions and lysats on the different paper pieces and took photos with the optimal filter combination for GFP and mRFP
- it was possible to detect fluorescence up to a dilution of 1:10 respectively 1:20
- Design of a modell of a box, which should be realized with an 3D- printer
- we had to find out which dimensions our box should have
- we measured the the size of a smartphone (samsung galaxy s5 mini) as sample for our prototyp
- we designed a box with a changable top, so the box can be adapted to every smartphone
- also we wanted a drawer, so the test strip can be placed there and inserted in the box
- we made some draft. These drafts were realized from Marco Radukic,so the box can be realized in a 3D printer
- Thomas Schäfer finally realized the box with his 3D printer
- The required devices were created by Standard BioBrick Assembly (Suffix Insertion).
- The positive clones were sequenced.
- The positive clones (confirmed by sequencing) were used for the measurement.
iGEM 2015 Measurement Interlab Study: