Team:UNC-Chapel Hill/Protocols

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PROTOCOLS

PCR amplification of MLC PCR amplification was performed using a TECHNE 3Prime thermocycler, the forward primer 5’ ACC GAC TCT CCG TTG CGT TAA G 3’and reverse primer 5’ TAA GGG TCA CTG GGC GAT ATT CG 3’. Both of primers had a melting temperature of approximately 59°C according to the OLIGOANYLYZER 3.1 on IDT’s website.

Transformation E. coli cells were thawed in a heat bath at 42 °C. A 20 μL volume of cells was pipetted into clean autoclaved 2.7 microtubes. Next, 2 μL of DNA was added (plasmids from kit, or ligation mixtures) and mixed in tubes by inverting. The mixture was then incubated on ice for half an hour. Cells were then heat shocked for 30 seconds and then placed on ice again for two minutes. 500 μL of SOC was added to the cells and placed in a shaker at 37 °C for one hour. Then the cells were poured onto the LB plates that had the corresponding antibiotic for the vector. The cells were then grown overnight (16 hours) in an incubator at 37 °C.

Digestion for Mlc and Backbone The Mlc parts were digested with 1 μL of 10 μM EcoRI, 1 μL of 10 μM SpeI, 3 μL of 10X 2.1 NEB buffer, 11 μL of USPI H2O, and 14 μL of the Mlc DNA. The backbone vector (BBa_pSB1C3) was digested with 1 μL of 10 μM EcoRI, 1 μL of 10 μM SpeI, 3 μL of 10X 2.1 NEB buffer, 1 μL of CIP, 19 μL of USPI H2O, and 5 μL of the backbone plasmid. The digestion mixtures were placed into the 37 °C incubator for over two hours.

Digestion for Inducible/Repressible and respective chromoproteins The inducible and repressible promoters were digested with 1 μL of 10 μM PstI, 1 μL of 10 μM SpeI, 3 μL of 10X 2.1 NEB buffer, 1 μL of CIP, 18 μL of USPI H2O, and 6 μL of the promoter DNA. The chromoprotein vector (BBa_K1073021 and BBa_K1073023) was digested with 1 μL of 10 μM PstI, 1 μL of 10 μM XbaI, 3 μL of 10X 2.1 NEB buffer, 19 μL of USPI H2O, and 6 μL of the chromoprotein plasmid. The digestion mixtures were placed into the 37 °C incubator for over two hours.

Digestion for Mlc and chromoproteins The Mlc parts were digested with 1 μL of 10 μM PstI, 1 μL of 10 μM SpeI, 3 μL of 10X 2.1 NEB buffer, 1 μL of CIP 14 μL of USPI H2O, and 10 μL of the Mlc DNA. The chromoprotein vector (BBa_K1073025) was digested with 1 μL of 10 μM PstI, 1 μL of 10 μM XbaI, 3 μL of10X 2.1 NEB buffer, 19 μL of USPI H2O, and 6 μL of the chromoprotein plasmid. The digestion mixtures were placed into the 37 °C incubator for over two hours.

DNA clean and Concentrate The purpose of the DNA clean and Concentrator kit from Zymo Research is to concentrate the DNA as well as purify the DNA of the enzymes and buffer used during the digestion reaction so that they will not interfere with the ligation reaction which comes after. The first step is to add 2X volume of DNA Binding Buffer and then load mixture into a Zymo-Spin Column and centrifuge at 12000 x g for 30 seconds. Next, 200 μL of DNA Wash Buffer is added and centrifuged. The wash step is repeated. Lastly, the column is placed into a new 2.7 mL microtube and eluted with 10 μL of water by centrifuging for 1 minute.

Ligation All ligation mixtures were 10 μL in total. Each had 1 μL of T4 DNA ligase and T4 DNA ligase 10X buffer. DNA amounts added varied from 0.5 μL to 4 μL in each individual ligation reaction depending on the concentration of the digestion mixture after the DNA clean and concentrate step. USPI H2O was added until a final volume of 10 μL. The ligation reactions were incubated at room temperature over night before transforming.

Liquid Culture Using a 200 μL pipet tip, colonies were picked from the plates and then dropped into clean glass tubes that have 5 mL of LB with the appropriate antibiotic. The cultures were then incubated at 37°C and shaken at 250 rpm for 16-24 hours

Glycerol Stock Into a 2.7 mL microtube, 600 μL of liquid culture and 400 μL of 50% glycerol is added. Then the tube is stored in a -40 °C freezer.

MiniPrep The purpose of the Miniprep is to extract the DNA from the cells. This was accomplished using a ZR Plasmid MINIprep Kit-Classic by ZYMO RESEARCH Company. First the DNA from the liquid culture was pelleted into a 2.7 mL microtube. Then 200 μL of P1 Buffer was added to resuspend the pellet. Then 200 μL of P2 Buffer was added and mixed thoroughly. The mixture was incubated at room temperature for 1 to 2 minutes. Next, 400 μL of P3 buffer was added and mix thoroughly. The mixture was centrifuged at 12000 x g for 2 minutes and the supernatant was added to the ZYMO-spinTM IIN column. The mixture was then centrifuged for 30 seconds into a collection tube. 200 μL of Endo-Wash Buffer was added and then centrifuged. Then 400 μL of Plasmid Wash Buffer was added and centrifuged for one minute. Lastly, the column was placed into a new 2.7 mL microtube and 30 μL of water was added and allowed to incubate for 5 minutes before centrifuging for one minute.

Diagnostic Gel The agarose gel is made by dissolving 1 gram of agarose in 100 mL of 1X TAE buffer. Then 1 μL of ethidium bromide is added to the solution. The solution is poured into the molding and cooled until it forms a gel (a comb is added to create the wells). Once the gel is cooled, it is reoriented so that the wells are on the negative side of the apparatus. 1X TAE buffer is added to the external wells so that it slightly overflows and covers the gel. The gel is now ready for DNA. The DNA in this experimental design were all cut with EcoRI and PstI, combined with DNA loading dye (1 to 5 ratio) and inserted into the gel. The gels were run for one hour at 120 mV, then analyzed under a High Performance UV Transilluminator made by the company UVP, which also has a camera attached for taking pictures.

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