Team:Charlottesville RS/Notebook/material

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Latest revision as of 15:09, 20 June 2014

Methods

Preparing competent cells

The bacteria were grown to log (or exponential) phase and 10mL of bacteria were aliquoted into 15 mL sterile plastic tubes. cells were pelleted using a tabletop centrifuge at 7000 x g for 10 minutes, or ~4000 rpm. The supernatant was poured off into liquid waste container. the tube was tapped to loosen the cell pellet, and d 5 mL of cold CaCL2 solution were added to the pellet. The cell solution was incubated on ice for 20 minutes. After incubation, the cells were spun for 5 minutes at 5000 x g (2500 rpm). The supernatant was poured off into the liquid waste container. One mL of cold CaCl2 solution was added to the cells and the cells were resuspended.

Rehydration of RFP and Alcohol Acetyltransferase I

Ten uL of dH2O were pipetted into the well of the corresponding Biobrick-standard part. This solution was pipetted up and down, then it was left to sit for 5 minutes. Five uL of the re-suspended DNA was transformed into competent cells. The transformed cells were plated, and were grown overnight.

Transformation of RFP and Alcohol Acetyltransferase I

The tubes were labeled as Transformation Control, Ligation: New Part, and Ligation Control. Five uL of RFP Control DNA was added to Transformation Control tube. Two uL of New Part ligation product was added to Ligation: New Part tube. Two uL of the RFP Control ligation product was added to the Ligation Control tube. The tubes were placed on ice to pre-chill. One tube of previously prepared competent cells was thawed and left on ice. Fifty uL of competent cells were pipetted into each of the 2.0mL micro-centrifuge tubes. The DNA and cell mixtures were incubated on ice for 30 minutes. During incubation, the water bath was heated to 42˚C. The tubes were placed in the waterbath for 60 seconds, then were incubated on ice again for 5 minutes. Two hundred uL of SOC media was added to each tube. The tubes were incubated at 37˚C for 2 hours. Two hundred uL of each tube was pipetted onto respective plates. The liquid was spread on the surface of the plate surface using glass beads. The plates were incubated lates face down at 37˚C for 12-14 hours.

Extraction of RFP and Alcohol Acetyltransferase I

Two culture tubes were spun down for 3 minutes at 8000 rpm. The supernantant was poured into a biological waste container. 250uL of Buffer P1 was pipetted into each 14mL culture tube, and the pellet was resuspended. The 1.7mL micro-centrifuged tubes were labeled with their respective part names, and transferred the parts were resuspended in it. 250uL of Buffer P2 was pippeted into each micro-centrifuge tube. The tubes were flipped to mix the solution. 350uL of Buffer N3 was pippeted into each tube.The tubes were flipped to mix the solution. The tubes in the micro-centrifuge were spun down for 10 minutes at 13,000 rpm. The spin columns were labeled for each tube, and the supernatant was pipetted to their respective spin columns. The spin columns were spun down at 13,000 rpm for 1 minute. The flow through was poured into the chemical waste beaker. 500uL was pipetted of Buffer PB into each of the spin columns. The spin columns were each spun down at 13,000 rpm for 1 minute. The flow through was poured into the chemical waste beaker. 750uL was pipetted of Buffer PE to each spin column. The spin columns were spun down at 13,000 rpm for 1 minute. The flow through was poured into the chemical waste beaker. The spin columns were spun down at 13,000 rpm for 1 minute to remove the remaining buffer. The spin columns were labeled “sterile 1.7mL micro-centrifuge” tubes with their respective part names. The appropriate filter tube was transferred to their respective tubes. 50uL of dH2O was pipetted to each filter tube. The samples sat for 1 minute, then the tubes were spun down at 13,000 rpm for 1 minute. The micro-centrifuged tubes were stored at 20˚C.

Digestion of RFP and Alcohol Acetyltransferase I

NEB Buffer 2 and BSA were thawed in room temperature water. Four 0.6mL tubes: Part A, Part B, pSB1C3, and RFP Control. 500ng of DNA were added to the appropriate tube. Distilled water was added to the tubes for a total volume of 42.5uL in each tube. 5uL of Buffer 2 was pippetted into each tube. 0.5uL of BSA was pippetted into each tube. In Part A tube, 1 uL of EcoRI enzyme was added and 1uL of SpeI enzyme was also added. In Part B tube, 1uL of XbaI enzyme was added, and 1uL of PstI enzyme was also added. In the pSB1C3 tube, 1uL of EcoRI enzyme was added, and 1uL of PstI enzyme was also added. In the RFP Control tube, 1uL of EcoRI enzyme was added, 1uL of PstI enzyme was also added. The solution was mixed by pipetting up and down, and spinning it for 5 seconds in the micro-centrifuge to collect the mixture at the bottom of the tube. The restriction digests were incubated at 37˚C for 30 minutes, then at 80˚C for 20 minutes. They were then stored at -20˚C.

Ligation of RFP, Alcohol Acetyltransferase I, and PSB1C3

The T4 DNA Ligase Reaction Buffer was thawed. 0.6mL was labeled as New Part. 2uL of the pSB1C3 linearized plasmid backbone digest was added. 3.3uL was added from Part A digest. 3.9uL was added from Part B digest. 1uL of T4 DNA Ligase Reaction Buffer was added. 0.5uL of T4 DNA Ligase was also added. The solution was mixed by pipetting, and by being centrifuged for 5 seconds. Another 0.6mL tube was labeled as Ligation Control. 2uL from RFP Control digest was added. 6.5uL of distilled water was added. 1uL of T4 DNA Ligase Reaction Buffer was added. 0.5uL of T4 DNA Ligase was added. The solution was mixed by pipetting, and centrifuging for 5 seconds. Both tubes were incubated at 16˚C for 30 minutes, then at 80˚C for 20 minutes and then stored at -20˚C.

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 			<li><a href="https://2014hs.igem.org/Team:Charlottesville_RS/Project"><span><span>Overall Project</span></span></a></li>
-			<li><a href="https://2014hs.igem.org/Team:Charlottesville_RS/Notebook/material"><span><span>Material & Methods</span></span></a>
+			<li><a href="https://2014hs.igem.org/Team:Charlottesville_RS/Notebook/material"><span><span>Material & Methods</span></span></a></li>
 			<li><a href="https://2014hs.igem.org/Team:Charlottesville_RS/Project/Applications"><span><span>Applications</span></span></a></li>
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-			<a href="https://2014hs.igem.org/Team:Charlottesville_RS/Parts" style="color: white">Parts<!--[if gt IE 6]><!--></a><!--<![endif]-->
+			<a href="https://2014hs.igem.org/Team:Charlottesville_RS/Safety" style="color:white">Safety<!--[if gt IE 6]><!--></a><!--<![endif]-->
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-				<li><a href="https://2014hs.igem.org/Team:Charlottesville_RS/Parts"><span><span>Submitted Parts</span></span></a></li>
-				<li><a href="https://2014hs.igem.org/Team:Charlottesville_RS/Parts/Characterization"><span><span>Characterization</span></span></a></li>
-				</ul>
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 			<a href="https://2014hs.igem.org/Team:Charlottesville_RS/Notebook/Overview" style="color: white">Notebook<!--[if gt IE 6]><!--></a><!--<![endif]-->
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-==Material==
-===Buffers & Solution===
-===Kits===
-===Marker===
-===Enzymes===
-===Plasmidvectors===
-===Synthetic oligonucleotides===
-===Phages===
-===Bacteria===
-===Bacteria Growth Media===
 ==Methods==
-===Preparing chemically competent cells===
+===Preparing competent cells===
+The bacteria were grown to log (or exponential) phase and 10mL of bacteria were aliquoted into 15 mL sterile plastic tubes. cells were pelleted using a tabletop centrifuge at 7000 x g for 10 minutes, or ~4000 rpm. The supernatant was poured off into liquid waste container. the tube was tapped to loosen the cell pellet, and d 5 mL of cold CaCL2 solution were added to the pellet. The cell solution was incubated on ice for 20 minutes. After incubation, the cells were spun for 5 minutes at 5000 x g (2500 rpm). The supernatant was poured off into the liquid waste container. One mL of cold CaCl2 solution was added to the cells and the cells were resuspended.
-===Transformation of bacteria===
-===Isolation of plasmid DNA by alkaline lysis (mini and maxiprep)===
-===DNA amplification using polymerase chain reaction (PCR)===
-===Purification of DNA from PCR reactions===
-===Enzymatic hydrolysis of DNA by restriction enzymes===
-===Agarose gel electrophoresis for separating DNA===
-===Isolation of DNA fragments from an agarose gel===
-===Ligation of dsDNA fragments===
-T4 DNA ligase was used to form covalent phosphodiesterbonds between doublestranded DNA fragments having blunt or compatible sticky ends. For the ligation the restricted vector -DNA and the insert were mixed 1:3 or 1:5 in a total ligationvolume of 20 µl. This mixture was incubated in ligation buffer using 5 Weiss units T4 DNA ligase over night at 16 °C or at room temperature for 20 minutes and afterwards used to transform chemical competent cells.
-[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
-===Lysing of bacteria by ultrasonication===
-For the lysis of bacteria an ultrasonic tip was used. The 10-15 ml of bacteria over night cultures were sonicated with an ultrasonic tip three time for 15 seconds. After sonication cell extract was observed under microscope.
-[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
-===Glycerol stock===
-To store bacteria for long term glycerol stocks were used. Therefore 1 ml of an over night culture were added to 150 µl of 80 % Glycerol into a cryo tube, vortexed and incubated at room temperature for 30 min. Afterwards the glycerole stock was stored at -80 °C.
-[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
-===Preparation of plating bacteria for bacteriophage experiments===
-A overnight culture of the appropriate ''E. coli'' strain was grown in LB medium containing 10 mM MgSO<sub>4</sub> and 0.2 % maltose at 30 °C to reduce the amount of cell debris in the medium. The added maltose leads to a substantial induction of the maltose operon including the lamb gene, which encodes the cell surface receptor to which bacteriophage λ binds. After harvesting the cells they were resuspended in 10 mM MgSO<sub>4</sub> and diluted to a final concentration of 2.0 OD<sub>600</sub>. The suspension of plating bacteria was stored at 4 °C for up to 1 week.
-[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
-===Bacteriophage λ plaque assay===
-Tenfold serial dilutions of the bacteriophage stocks were prepared, 100 µl of it mixed with the same amount of plating bacteria, incubated for 20 minutes at 37 °C to allow the bacteriophage particles to adsorb to the bacteria, this mixture added to 3 ml molten top agar which was kept liquid at 48 °C and the entire contend poured onto a agar plate. After harden of the top agar the inverted plates were incubated at 37 °C over night. On the next day plaques could be counted.
-[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
-===Preparing Stocks of Bacteriophage λ===
-Completely lysed agarose plates were prepared by mixing 0.1 ml plating bacteria with ~10<sup>5</sup> pfu, incubation for 20 minutes at 37 °C and that bringing out the mixture in 3 ml molten top agarose. Agarose is preferred in this protocol in order to minimize contaminations with polyanionic inhibitors that can interfere with subsequent enzymatic analysis of the bacteriophage DNA. The plates were incubated 12-16 hours without inversion at 37 °C. Plates were not inverted during incubation to encourage sweating of fluid onto the surface of the dish, which allows the bacteriophage to spread more easily. The plates were than overlayed with 5 ml SM and incubated while shaking at 4 °C over night. The SM was that transferred to a tube and the plate washed again with 1 ml SM. 2 % v/v chloroform was added and bacterial debris removed by centrifugation at 10,000 g for 10 minutes at 4 °C. The concentration of infectious particles in the stock was measured by plaque assays as described above.
-[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
-===Preparing of λ DNA===
-Lambda DNA was prepared using the QIAGEN® Lambda mini kit following the manufacture instructions.
-[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
-===In vitro packaging of λ DNA===
-In vitro packaging was conducted using the MaxPlax lambda packaging extracts from Epicentre Biotechnologies following the manufacture instructions.
-[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
-===Colicin Activity Test===
-To measure the killing efficiency and which amount of cells or colicins are needed to reach any killing activity a colicin activity test was carried out. Therefore bacteria containing the colicin plasmid (''E. coli'' TOP10 or ''E. coli'' MG1655) and GFP producing cells (reference promoter [http://partsregistry.org/wiki/index.php?title=Part:BBa_I20260 BBa_I20260], ''E. coli'' TOP10) were inoculated in TB-media with appropriated antibiotics at 37 °C for 4 to 6 hours and the optical densities of the two strains were adapted. The colicin cells themselves, their produced supernatant or the supernatant of the lysed colicin cells were added in different ratios to a constant amount of GFP producing cells. The total volume was kept constant and the missing amount added with TB-media without antibiotics. The colicin production was induced by several concentrations (0 M-100 nM) of N-Acyl-Homoserin-Lactone (AHL). The OD and GFP intensities were measured at 37 °C in the Tecan Microplate Reader every half an hour for about 12 hours.
-As a negative control the similar test was carried out with cells, containing the same plasmid without the colicin operon on it.
-[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
-===Lysis test===
-Lysis of killercells dependent on the AHL concentration was measured due to a lysis test. Therefore ''E. coli'' TOP 10 containing the luxR-colicinE1 part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K150009 BBa_K150009]) and a GFP producing part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I20260 BBa_I20260]) were inoculated in TB-media with appropriate antibiotics at 37 °C for 4 to 6 hours. As reference ''E. coli'' TOP 10 cells containing a luxR part (comparable to [http://partsregistry.org/wiki/index.php?title=Part:BBa_F2620 BBa_F2620]) instead of the luxR-colicinE1 part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K150009 BBa_K150009]). For the lysis measurement 250 µl of the liquid cultures were diluted with 250 µl TB-media. The Lux pR promoter was activated with AHL concentrations from 0 M up to 50 nM. The optical densities and GFP intensities were measured in Tecan Microplate Reader every half-hourly for about 12 hours.
-[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
-===Sender Activity Test===
-The sender activity test was performed to quantify the AHL production of the cells containing the sender part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K150000 BBa_K150000]). Therefore TB-media with the appropriated antibiotic was inoculated with 2 µl/ml of an overnight culture. For 7 hours every hour the OD of the medium was measured and 5 ml of the culture were spin down to produce supernatant. The sterile filtered supernatant, still containing the AHL, was stored at 4 °C. The supernatant of every hour was then added in different ratios to a constant amount of AHL inducible cells which produce GFP after induction. The OD and GFP intensities were measured at 37 °C in the Tecan Microplate Reader every half an hour for about 12 - 24 hours. The same test was carried out with amplifier cells ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I15030 BBa_I15030]). To calculate the produced amount of AHL by sender or amplifier cells as reference the test were performed also with different AHL concentration (0 M-100 nM) instead of AHL producing cells.
-[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
-===LuxS Activity Test===
-This protocol for the functional check AI-2 production is mainly adapted from a paper of Vanderleyden et al. [1] , for further questions please refer to it. 10ml ONC DH5α strain with LuxS (S) and 10ml DH5α strain with ptrc99α as blank (B) were incubated in LB to a volume of 50mL each at 37°C up to an OD of 0.35 in 100ml erlenmeyer flasks. Then S and B each were induced with 500 µM IPTG for 3h, following centrifugation at maximum speed for 20min. The supernatants were then transferred in two new falcons by pipetting or decanting. Optional the supernatant was sterile filtrated and/or 10 g/L NaCl was added to a final concentration of ~20g/L. 10 mL ONC of ''V. harveyi'' reporter (BB120 in LB plus at 30 °C) was incubated in LB plus to a final volume of 50 mL at 37°C up to an OD of 0.35 in a 100 mL erlenmeyer flask. Supernatant S and B were both mixed with ''Vibrio'' cells at ratios 1:3, 1:5 and 1:10 each to a final volume of 5 mL in 50 mL falcon tubes and incubated for 30min at 30°C. Subsequently the cells were incubated again for 3h, OD and Luminescence were measured with a microplate reader. [[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
-[1] Sigrid C. J. DeKeersmaecker and Jos Vanderleyden ‘Constraints on detection of autoinducer-2 (AI-2) signalling molecules using Vibrio harveyi as a reporter’, Microbiology 2003 149: 1953-1956.
-===Conjugation test===
-Equal amounts of donor (cells harbouring the oriT containing plasmid and pUB307) and acceptor cells from overnight cultures were centrifuged (2 min, 13000 rpm), the pellet washed twice with 1.5 ml LB medium without antibiotics, the pellets combined and this mixture resuspended in 100 µl LB medium without antibiotics. This cell mix was transferred to a filter membrane on a LB agar plate and incubated here for a time range from 0 to 42 minutes in 6 minutes steps at 37 °C. The membrane was then transferred in 1 ml LB medium, vortexed, the medium diluted and plated out on LB agar plates with proper antibiotics for plasmid harbouring the oriT and antibiotic resistance of acceptor bacteria. On the next day the number of transconjugants was counted and therewith the conjugation efficiency calculated as ratio of cell density of transconjugants to cell density of donor cells depending on the reaction time.
-[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
-=== Receptor Polarization Test ===
-To check the functionality of the fusion receptor constructs each construct, on pDK48, was transformed into strain UU1250 together with pDK36 (YFP-CheA). The expression of the receptor was induced with 0.01% arabinose, of YFP-CheA with 30µM IPTG. Polarization was tested by putting 10 µL of the cells on a 0.5% agarose pad and making snapshots using a Zeiss Axio Imager.Z1 fluorescent microscope.
-===Swarm Assays===
-The sensitivity of the LuxP-tar fusion constructs to AI-2 was tested in swarm assays. For this two different strains were used: ''E. coli'' UU1250 and ''E. coli'' HCB33. The assays were performed in TB medium for UU1250 and TB or minimal medium for HCB33 (0.3% agar). As chemo attractant either 3 µl of an AI-2 producing ONC, induced with 100 nM IPTG, or 10 µl supernatant from those cells was spotted 13 times on the plate to create a center line. Gradient building was allowed for 12-16 hours at 4 °C. Afterwards the fusion receptor expressing cells (0.01% arabinose induced overnight) as well as non-chemotactic reference strains were spotted at a distance of about 3-3.5 cm to the attractant line. Swarm plates were incubated at 30 °C and pictures taken after 24, 48 and 72 hours.
-[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
-===Microscopy – Chemotaxis===
-An Applied Precision - DeltaVision wide field microscope was used to visualize chemotactic behaviour of the LuxP-tar fusion. ''E. coli'' UU1250 and ''E. coli'' HCB33 were used for these measurements. The monitoring was performed with Labtek<sup>TM</sup> chambered coverglass systems (Nunc) which were filled with 2 ml of TB media, Tethering Buffer and minimal media containing 0.2 % agar. As chemo attractant either 3 µl of an AI-2 producing ONC, induced with 100 nM IPTG, or 10 µl supernatant from those cells was spotted at the one side of the chamber. Gradient building was allowed for 12-16 hours at 4 °C.
-Afterwards either fusion receptor expressing cells (0.01% arabinose induced overnight) or non-chemotactic reference strains were spotted at the center of the chamber. The chamber was then incubated for several hours at 30 °C. After 3 hours frame pictures were taken, using confocal fluorescence imaging and 8x6 frames. (8 pictures in length, 6 in height)
-Images were processed and auto arranged with housemade ImageJ plugins. Matlab was used for the quantitative analysis.
-[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
-===Microscopy – Colicin Toxicity===
-The killing efficiency of colicin producing cells was monitored using an Applied Precision - DeltaVision wide field microscope. Therefore a prey and a killer strain were designed in ''E. coli'' TOP10. The prey strain contained a GFP producing part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I20260 BBa_I20260]) as well as the luxI sender part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K150000 BBa_K150000]) and the killer strain contained as marker a mCherry producing part as well as the LuxR-colicinE1-receiver ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K150009 BBa_K150009]).
-Measurements were prepared with 1 ml of a prey ONC and 1 ml of a killer ONC in 3 ml TB media containing 0,01% arabinose and 10 nM AHL for induction of colicin production. A second probe in which cells without colicin genes were used instead of the killer cells was prepared in parallel. Both samples were incubated for 6-7 hours at 37 °C.
-Monitoring was performed on the Deltavision microscope with 8-well Labtek<sup>TM</sup> chambered coverglass systems (Nunc). For that 6 µl of each sample were distributed equaly in 200 µl TB media. Frame pictures have been taken using 8x8 frames evenly distributed over the whole chamber.
-Images were processed and auto arranged with housemade ImageJ plugins.
-[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
-===Microscopy – Cell lysis===
-To visualize the lysis of prey and killer cells cultures were observed on Zeiss Axio Imager.Z1 microscope. The killer strain (''E. coli'' TOP 10) contained the luxR-colicinE1 part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K150009 BBa_K150009]) and a mCherry producing part and the prey strain contained the senderpart ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K150000 BBa_K150000]) and a GFP producing part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I20260 BBa_I20260]). Both strains were inoculated in TB-media with appropriate antibiotics at 37 °C for 4 to 6 hours. Thereafter OD was adjusted and the killer strain was induced by 25 nM AHL. After 2 hour 200 µl of the induced killer culture and 200 µl of the prey culture were combined and centrifuged down. For microscopy pelleted cells were diluted with 10 µl and plated on a 0.5% agarosepad. Fixed positions were observed half hourly in the mCherry and GFP channel with a 100x oil immersion objective.
-[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
-===Cell Culture===
-MCF-7 cells (breast cancer cell line) wt or stably expressing cytoplasmic GFP, were plated in 24 well plates (150 thousand cells per well) with 10 % FCS (fetal calf serum) DMEM (Dulbecco's Modified Eagle Medium) and incubated overnight at 37 °C, 5 % CO2.
+===Rehydration of RFP and Alcohol Acetyltransferase I===
-E. coli TOP 10 strains containing LuxR-colicin E1-receiver (BBa_K150009) and strains containing LuxR-receiver (no colicin) were grown overnight in TB media with appropriate antibiotics at 37 °C to an OD of 1.0 and diluted in the morning to 0.1. Once bacterial cells reached an OD of 0.35 they were harvested and inoculated on the MCF-7 wt and on MCF-7-GFP cells.
+Ten uL of dH2O were pipetted into the well of the corresponding Biobrick-standard part. This solution was pipetted up and down, then it was left  to sit  for 5 minutes.  Five uL of the re-suspended DNA was transformed into  competent cells. The transformed cells were plated, and were grown overnight.
-[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
-===Eucaryotic Killing Assay with Colicins===
+===Transformation of RFP and Alcohol Acetyltransferase I===
+The tubes were labeled as ''Transformation Control'', ''Ligation: New Part'', and ''Ligation Control''. Five uL of ''RFP Control DNA'' was added to ''Transformation Control'' tube. Two uL of ''New Part'' ligation product was added to ''Ligation: New Part'' tube. Two uL of the ''RFP Control'' ligation product was added to the ''Ligation Control'' tube.  The tubes were placed on ice to pre-chill. One tube of previously prepared competent cells was thawed and left on ice.  Fifty uL of competent cells were pipetted into each of the 2.0mL micro-centrifuge tubes.  The DNA and cell mixtures were incubated on ice for 30 minutes. During incubation, the water bath was heated to 42˚C. The tubes were placed in the waterbath for 60 seconds, then were incubated on ice again for 5 minutes.  Two hundred uL of SOC media was added to each tube.  The tubes were incubated at 37˚C for 2 hours.  Two hundred uL of each tube was pipetted onto respective plates. The liquid was spread on the surface of the plate  surface using glass beads. The plates were incubated lates face down at  37˚C for 12-14 hours.
-Once MCF-7 cells were confluent (12h after passage), E. coli TOP 10 bacteria were spinned down (1 ml of OD = 0.35) and resuspended with DMEM medium from the original wells containing the MCF-7 wt/MCF-7-GFP cells. A 24-well plate was used containing MCF-7  cells stained with Propidium Iodide (PI) alone, with LuxR-receiver cells not containing colicin and LuxR-receiver-colicin E1 cells. The second 24-well plate with MCF-7GFP was processed as described for wt MCF-7, but without any PI staining. AHL was added to every well to a final concentration of 5 nM. Well content was collected at different time-points (0 – 6 h) and when necessary, PI was added to the suspension 15 minutes before Flow Cytometer analysis.
+===Extraction of RFP and Alcohol Acetyltransferase I===
-[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
+Two culture tubes were spun down for 3 minutes at 8000 rpm. The supernantant was poured into a biological waste container. 250uL of Buffer P1 was pipetted into each 14mL culture tube, and the pellet was resuspended.  The 1.7mL micro-centrifuged tubes were labeled with their respective part names, and transferred the parts were resuspended in it. 250uL of Buffer P2 was pippeted into each micro-centrifuge tube. The tubes were flipped to mix the solution. 350uL of Buffer N3 was pippeted into each tube.The tubes were flipped to mix the solution. The tubes in the micro-centrifuge were spun down for 10 minutes at 13,000 rpm. The spin columns were labeled for each tube, and the supernatant was pipetted to their respective spin columns. The spin columns were spun down at 13,000 rpm for 1 minute. The flow through was poured into the chemical waste beaker. 500uL was pipetted of Buffer PB into each of the spin columns. The spin columns were each spun down at 13,000 rpm for 1 minute. The flow through was poured into the chemical waste beaker. 750uL was pipetted of Buffer PE to each spin column. The spin columns were spun down at 13,000 rpm for 1 minute. The flow through was poured into the chemical waste beaker. The spin columns were spun down at 13,000 rpm for 1 minute to remove the remaining buffer. The spin columns were labeled “sterile 1.7mL micro-centrifuge” tubes with their respective part names. The appropriate filter tube was transferred to their respective tubes. 50uL of dH2O was pipetted to each filter tube. The samples sat for 1 minute, then the tubes were spun down at 13,000 rpm for 1 minute. The micro-centrifuged tubes were stored at 20˚C.
-===Flow Cytometer Acquisition===
+===Digestion of RFP and Alcohol Acetyltransferase I===
+NEB Buffer 2 and BSA  were thawed in room temperature water. Four 0.6mL tubes: ''Part A'', ''Part B'', ''pSB1C3'', and ''RFP Control''. 500ng of DNA were added to the appropriate tube. Distilled water was added to the tubes for a total volume of 42.5uL in each tube. 5uL of Buffer 2 was pippetted into each tube. 0.5uL of BSA was pippetted into each tube. In ''Part A'' tube, 1 uL of EcoRI enzyme was added and 1uL of SpeI enzyme was also added. In ''Part B'' tube, 1uL of XbaI enzyme was added, and 1uL of PstI enzyme was also added. In the ''pSB1C3'' tube, 1uL of EcoRI enzyme  was added, and 1uL of PstI enzyme was also added. In the ''RFP Control'' tube, 1uL of EcoRI enzyme was added, 1uL of PstI enzyme was also added. The solution was mixed by pipetting up and down, and spinning it for 5 seconds in the micro-centrifuge to collect the mixture at the bottom of the tube. The restriction digests were incubated at 37˚C for 30 minutes, then at 80˚C for 20 minutes. They were then stored at -20˚C.
-At the referred timepoints, supernatants were collected and spinned down. Eukaryotic cells were detached by trypsinization (200 μl) for 10 minutes at 37 °C and then resuspended in 800 μl of KH + 1 % BSA (Krebs Henseleit medium with 1 % Bovine Serum Albumin). After resuspending, well content was added to the precipitated supernatant to account for floating dead cells. PI was added to each sample (1:1000) and incubated at RT for 15 minutes before acquisition. PI positive cells and GFP expressing cells (%) were measured for 3000 cells for each condition. Histograms and fluorescent measurements were obtained using a Cytomics FC 500 MPL from Beckman&Coulter.
+===Ligation of RFP, Alcohol Acetyltransferase I, and PSB1C3===
-[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
+The T4 DNA Ligase Reaction Buffer was thawed. 0.6mL was labeled as ''New Part''. 2uL of the ''pSB1C3'' linearized plasmid backbone digest was added. 3.3uL was added from ''Part A'' digest. 3.9uL was added from ''Part B'' digest. 1uL of T4 DNA Ligase Reaction Buffer was added. 0.5uL of T4 DNA Ligase was also added. The solution was mixed by pipetting, and by being centrifuged for 5 seconds. Another 0.6mL tube was labeled as ''Ligation Control''. 2uL from ''RFP Control'' digest was added. 6.5uL of distilled water was added. 1uL of T4 DNA Ligase Reaction Buffer was added. 0.5uL of T4 DNA Ligase was added. The solution was mixed by pipetting, and centrifuging for 5 seconds. Both tubes were incubated at 16˚C for 30 minutes, then at 80˚C for 20 minutes and then stored at -20˚C.