Team:NC School of Sci Math/Lab Notebook

From 2013hs.igem.org

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* We should be able to figure out the others soon
* We should be able to figure out the others soon
* 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.
 
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<br/><br/>
 
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<b>The PCR program was done as follows:</b><br/>
 
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94°C/3min||94°C/30s|60°C/30s|72°C/3min 45s||30x 72°C/10min|4°C/forever
 
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Afterwards, 3 µl PCR product were loaded onto a 1 % agarose gel.
 
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<h4><a style="color:#168dc4;" name="05/28/2012">05/28/2012</a></h4>
 
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<b>Transferring the parts into standard registry plasmid pSB1C3:</b>
 
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*Restriction digest of 1 µg of each sample and of the standard backbone
 
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*After 30 min SapI is added to the backbone digest
 
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*After overall 60 min Qiagen nucleotide removal kit was used to purify DNA
 
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*heat inactivation of remaining enzymes at 80°C
 
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*concentration measurement with nanodrop
 
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*Ligation
 
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<br/>
 
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*heat inactivation at 70°C
 
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<h4><a style="color:#168dc4;" name="06/02/2012-con">06/02/2012</a></h4>
 
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<b>Inoculate:</b><br/><br/>
 
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<u>Protocol</u>:  Constructs grown as overnightculture on agarplates:
 
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<br/>
 
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* #5  <html><a href="http://partsregistry.org/Part:BBa_K862000"> precA_LacZ</a></html>
 
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* #7  <html><a href="http://partsregistry.org/Part:BBa_K862001"> psulA_LacZ</a></html>
 
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* #8  <html><a href="http://partsregistry.org/Part:BBa_K862002"> precB_LacZ</a></html>
 
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* #9 precC_LacZ<br/>
 
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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/>
 
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<br/>
 
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<table style="margin:15px;border-collapse:collapse">
 
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<tr><td style="padding:5px;border:2px solid black">Part</td><td style="padding:5px;border:2px solid black">DNA concentration</td></tr>
 
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<tr><td style="padding:5px;border:1px solid black">#5.1</td><td style="padding:5px;border:1px solid black">16 ng/µl</td></tr>
 
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<tr><td style="padding:5px;border:1px solid black">#5.2</td><td style="padding:5px;border:1px solid black">6 ng/µl</td></tr>
 
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<tr><td style="padding:5px;border:1px solid black">#7.1</td><td style="padding:5px;border:1px solid black">46 ng/µl</td></tr>
 
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<tr><td style="padding:5px;border:1px solid black">#7.2</td><td style="padding:5px;border:1px solid black">57 ng/µl</td></tr>
 
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<tr><td style="padding:5px;border:1px solid black">#8.1</td><td style="padding:5px;border:1px solid black">84 ng/µl</td></tr>
 
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<tr><td style="padding:5px;border:1px solid black">#8.2</td><td style="padding:5px;border:1px solid black">91 ng/µl</td></tr>
 
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<h4><a style="color:#168dc4;" name="06/07/2012">06/07/2012</a></h4>
 
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<p><b>Three parts were sent to the parts registry</b>:
 
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You can find all BioBricks under: <a href="http://2012HS.igem.org/Team:Heidelberg_LSL/Parts"> Parts </a> </p>
 
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Transformation of <i>E.coli</i> strain BL21 (DE3) with our constructs the samples were plated on LB agarplates with Ampicillin.
 
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<h4><a style="color:#130ed2;" name="04/28/2012">04/28/2012</a></h4>
 
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Inoculation of overnight cultures in LB liquid medium with Amp from the plates.
 
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<h4><a style="color:#130ed2;" name="04/29/2012">04/29/2012</a></h4>
 
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<b> Experiment 1: </b><br/>
 
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*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.
 
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<center><img src="https://static.igem.org/mediawiki/2012hs/8/80/Bildschirmfoto_2012-06-11_um_23.47.05.png" width="500"/> </center>
 
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<h4><a style="color:#130ed2;" name="05/27/2012">05/27/2012</a></h4>
 
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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
 
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<img src="https://static.igem.org/mediawiki/2012hs/7/7f/Bildschirmfoto_2012-06-12_um_00.03.28.png" width="635"/></html>
 
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<br/>
 
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<b>Fig. 1: Picture of the color gradient visible in the X-Gal assays with different exposure times</b>
 
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<b>Testing of the precA_GFP construct</b><br/>
 
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*Overnight culture of precA_GFP was diluted 1:5
 
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*3ml of the culture were induced by UV radiation in the gel chamber for 30min
 
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*The induced culture and 3ml of uninduced culture were incubated for 30min at 37°C / 80rpm
 
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*After the incubation both cultures were compared using the fluorescence microscope
 
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<img src="https://static.igem.org/mediawiki/2012hs/6/6f/GFP_Measurement.PNG"  width="635"/><br/>
 
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<b>Fig. 2: Results of the GFP samples underneath fluorescence microscope</b>
 
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<h4><a style="color:#130ed2;" name="05/28/2012-cal">05/28/2012</a></h4>
 
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<b> Testing under real life conditions: </b>
 
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*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
 
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<img src="https://static.igem.org/mediawiki/2012hs/f/f9/Outdoor-Test.png" width=635> </img>
 
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<h4><a style="color:#130ed2;" name="06/07/2012-cal">06/07/2012</a></h4>
 
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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/>
 
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<img src="https://static.igem.org/mediawiki/2012hs/c/c7/XGAL_Measurement.PNG" width="635" /><br/>
 
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<b>Fig. 1: All constructs in comparison</b>
 
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Revision as of 16:01, 18 June 2013

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2013


Planning and Development

01/11/2013

Brainstorming for project ideas

  • LDL-cholesterol biosensor
  • Ocean salinity regulator (especially for coral reefs)
  • Water contaminant multibiosensor


More research should be done

  • Find related projects that have been completed
  • Obstacles?
  • BioBricks?
  • Presentations in 3-4 weeks

01/18/2013

Discussions of each project

  • LDL-cholesterol biosensor
    • Could be used as a blood test
    • Blood sugar monitor-like device
    • Hard to measure any color change in blood
  • Ocean salinity regulator (especially for coral reefs)
    • Need a lot of bacteria
    • Environmental concerns
    • Bacteria can do well in coral reefs
  • Water contaminant multibiosensor
    • Several Biobrick promoters for common contaminants
    • Usage in sewer tanks
    • Many different colors of fluorescent proteins
    • Could incorporate newer technology for better color-sensing
    • Will look up data on promoter/FP expression

02/01/2013

Presentations

  • All ideas sound promising
  • Meet next week to decide which idea to pursue

02/08/2013

Decision on project

  • LDL-cholesterol idea won't work
    • Costly and hard to detect FPs in blood
  • Multibiosensor is appealing
    • Many applications
    • Expandable with many different FPs
    • Could bring in new technology
  • Ocean salinity regulator is difficult because of the ocean
    • Environmental factors and unknown variables

Multibiosensor chosen

02/15/2013

A few members met with Mr. Jon Davis to discuss potential technology

  • Suggested a Google device called ADK
    • Comes with a colorimeter
    • Can be programmed for many functions
    • School has a few we could try out

02/22/2013

Goals for the immediate future

  • Find Biobricks
  • Set up schedule for lab work
  • Further research into possible uses
  • Preliminary lab experiments


Project Proposal


03/01/2013

Discussion of upcoming project proposal:

  • Introduction, Solution Statement, and Methods
  • Need to find out some background information
  • Should make use of Tinkercell modeling

03/04/2013

Assignments of roles for the upcoming project proposal:

  • Madeline will investigate background information
  • Danny will work on Tinkercell models
  • Jack will write the solution statement and methods sections
  • Synthesize our writing on a Google Doc
  • Try to have individual parts done in about 2 weeks

03/18/2013

The following models have been created

  • We should use these in the proposal, but continue to work on the Tinkercell model

Design.jpg

03/25/2013

Everyone began editing the proposal on a Google Doc today

  • Want to be finished by March 29th
  • Tinkercells will probably not be ready in time
    • Having a lot of trouble getting fluorescent protein levels to zero when they should be
    • Danny will talk to Morgan to see if he can help
  • Edit Edit Edit!

03/27/2013

  • Proposal is coming along well
  • Need to finish the methods, but otherwise complete
  • Take pictures of the ADK and lab work

03/29/2013

  • Proposal is done!
  • Will submit soon

04/06/2013

Time to focus on creating the plasmid

  • Finished Tinkercell model will help a bunch
  • Do we want to think about ordering de novo from a gene synthesis company?
  • Computer modeling is the best approach for now

04/17/2013

  • Danny has a working Tinkercell model for the copper detector!
    • Outputs increased levels of TagRFP as ion concentration increases
  • We should be able to figure out the others soon
  • The lead detector may be especially difficult because the ion binds to a promoter to create a transcription factor

Calibration and Characterization


04/27/2012


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.

Screenshot.jpg


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.

PGLOGraphs.png

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.