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From 2014hs.igem.org

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.