* The lead detector may be especially difficult because the ion binds to a promoter to create a transcription factor
* The lead detector may be especially difficult because the ion binds to a promoter to create a transcription factor
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<b>Colony PCR:</b><br/>
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Screening of the two different biobrick clonings done by colony-PCR.
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<br/><br/>
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<b>Protocol:</b><br> A reaction mix was prepared containing 0.2 µl of each screening primer used for the screening, 9.6 µl of water and 10 µl of 2x PCR mastermix (fermentas). Finally, a colony was picked from a plate using a sterile pipette tip and was dipped into the PCR mix a few times. 12 clones were screened for each different cloning setup as follows:<br/>
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<br/>
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<u>precB_LacZ</u>: primers VF2: and precB Reverse: were used. Only in case precB was successfully cloned into the backbone containing the reporter gene, we would get a PCR product.
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<br/><br/>
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<u>precC_LacZ</u>: primers VF2 and VR (standard sequencing primers) were used. We compared the product size of the different clones to the product size from the original vectors from the registry (only containing precA or LacZ). In case the cloning was successful, we should see a 200 bp shift in product size, which can be detected by gel electrophoresis.
A reaction mix was prepared containing 20 ml LB medium and 20 µl Chloramphenicol (50mg/µl). Finally a single blue colony (except for #9) was picked from an agar plate using a sterile pipette tip and was dropped into a tube with 2 ml of the reaction mix.<br/>
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Except for #9 we picked two colonies of each plate and inoculated separately. For plate #9, we picked four white colonies to check for basal expression.<br/>
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After picking the colonies the tubes, in which the pipette tips where dropped, were put into 20 ml LB Medium .<br/>
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Several hours later a miniprep of the grown cultures with the Qiagen Kit followed. DNA was eluted in TE Buffer.<br/>
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Measurement of DNA concentration with Nanodrop:<br/>
*day culture was inoculated with 1:30 dilution of overnight culture in LB with Ampicillin
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*day culture was incubated for 3 h
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*measurement of optical density using a photometer at 600 nm to confirm bacterial growth
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*distribution of each 3 ml per sample to 6-well-plates
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*7 replicate plates one per time span: 0 s, 5 s, 10 s, 30 s, 5 min, 10 min of exposure time
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*Exposure to UV-light in the Intas gel IX imager with the above-mentioned time spans
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*Addition of 30 µl X-Gal (2mg/ ml) to samples #5, #7 (constructs containing LacZ) plates sealed with parafilm
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*Incubation at 37°C, 50 rpm for 45 min, visual color change: LacZ samples became blue
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*measurement of optical density at 600 nm of #5 and #7<br/>
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Problem: maximum absorption of X-Gal at 615-650 nm interferes with measurement of bacterial density at 600 nm – no change in absorption although blue colour was visible
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<br/><br/>
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<b> Experiment 2: </b><br/>
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*overnight cultures centrifuged at 4000 rpm for 7 min
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*pellet resuspended in 25 ml LB Amp
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*The bacterial suspension is again exposed to UV-light with time spans of 0s, 20s, 60s, 300s, 600s.
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*After the exposure X-Gal is added to the samples. Incubation at 37°C, 80 rpm
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*first visible color change after 5 min in #7, #5 with X-Gal
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*for a quantification the assays were plated out with duplicates on a 96w-Plate. LB medium was used as a blank reference. ONPG was added and the absorbance was measured with the plate reader
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*ONPG was used because its absorbance maximum differs from the wavelength you use to measure the optical density
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*after that we measured the optical density of our samples using the photometer to get the true expression.
Repetition of the second experiment at 29/04/2012.<br/>
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*overnight cultures centrifuged at 4000 rpm for 7 min
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*pellet resuspended in 25 ml LB Amp<br/>
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*The bacterial suspension is again exposed to UV-light with time spans of 10min 8min 6min 4min 2min 0min
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*After the exposure X-Gal is added to the samples. Incubation at 37°C, 80rpm
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<u>Experiment failed.</u><br/>
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No clear graduation between the samples of different exposure times was observed.<br/>
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<u>Possible causes</u>: big differences in incubation time after exposure<br/>
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<b>Improvements in 4. experiment:</b><br/>
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*24 wellplate recA and sulA
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*for 5 min in UV light then the first row was transfered to another well-plate. The rest went again for 5 min under UV light, then the second row, with now an time period of 10 min.
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*Same procedure for all the other rows. Time periods 5min 10min 15min 20min 30min and a controll sample with 0min
*culture of precA_LacZ and psulA_LacZ was centrifuged at 4000rpm for 8 min
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*pellet was resuspended in LB Amp
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*the samples were put into 2 6-well plates (3 ml/well)
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*plates were tightly sealed, desinfected and put outdoor either into the sun or shadow for 75 min
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*after 30 min incubation X-Gal was added to final concentration of 200 µg/ml the coloring of the samples were observed over time; intensity of the coloring in the wells were measured using the ImageJ software package
Overnight cultures precB_LacZ were diluted 1:2 with LB Medium and transferred onto 24-well-plates, 500 µl per well. Duplicates were exposed to UV-radiation in the UV-chamber for 5, 10, 15, 20 and 30 min. <br/>
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X-Gal stock solution was diluted 1:100.<br/>
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After 1h incubation 50µl X-Gal was added to final concentration of 200 µg/ml. The coloring of the samples were observed over time. Intensity of the coloring in the wells were measured using the imageJ software package.<br/>
Template:NotebookLower
/b>
4 ml bacterial culture is pelleted by centrifugation at 10.000 rpm for 1 min. Afterwards, the pellet is resuspended in 250 µl buffer P1. 250 µl buffer P2 are added and the culture is gently inverted and incubated for 5 min. 350 µl buffer N3 are added and the suspension is centrifuged at 13.000 rpm for 10 min. The supernatant is subsequently loaded onto a miniprep column and centrifuged for 1 min/max speed. Afterwards the column is washed with buffer PB (500 µl) and buffer PE (750 µl) by loading onto the column and subsequent centrifugation for 1 min/max speed. After a last centrifugation step for drying the column (again 1 min/max speed), 50 µl of water are added in order to dissolve the DNA again. After 1 min incubation, DNA is eluted by centrifugation for 1 min/max speed.
Lab Notebook
To begin our experiment, we needed to create a device that could detect color. We chose to use the Google ADK, an open ended platform that allows users to take control of many sensors that come attached to an Arduino board, including a colorimeter. However, a standard ADK is set up to match any color presented to it, using LED lights. In some of our preliminary tests, the reflection of the light from these LEDs skewed data. Therefore, we modified the code of the ADK, using Google's software developer package, so that it does not turn on its LED lights but instead outputs colorimeter data to a computer. An example of this output is shown below.
As a foray into our project, we first took some preliminary data using our modified Google ADK and pGLO E. coli expressing Green Fluorescent Protein. We found that we were able to detect a distinct change in color. Some baseline color was present from the UV Light source used for the experiment. The graphs below clearly show that our system is able to detect E. coli expressing GFP.
Small LED lights were used to test the ADK's ability to detect red, blue, and yellow lights. The results showed the same trends as the GFP test, indicating that we would be able to distinguish between several different colors expressed. In general, red, green, and blue lights show dramatic increases in the intensity of that color, while the other two colors remain about the same. Yellow light however, increase both red and green light, while blue remains at the same level. From these observations, we are able to identify which type of light is shown based only on the data collected.
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