IGEM:Hong Kong HKUST/Investigations/ Plasmid Cloning by Standard Assembly /Entry Base

Authors Chan Hei Wing Natalie Chan Lok Man Daniel Chow Edward Ng Abstract Introduction Methods and Material The promoter selected is BBa_R0010, which is repressed by the presence of lac I protein. However, there is only one lac operon in E.coli while plasmids with BBa_R0010 outnumber the operon. lac I protein produced is merely sufficient to repress some of the promoter. There are still active promoters and thus gene coding for mRFP will be expressed. The target fragment coding for mRFP is BBa_K516032, which contains RBS (BBa_B0032), mRFP CDS (BBa_1010) and double terminator (BBa_B0015). Transformation Following the modified protocol provided by NEB (Transformation Protocol, n.d.), the transformation was carried out in order to amplify the desired DNA. Specially, bacteria with BBa_R0010 and BBa_K516032 were expected to harvested from this step. For each target DNA fragment, 2 μL of DNA from kit plate was used. For ligation product, 10 μL of reaction mixture were used. 1 minute and 30 seconds of heat shock was used. Inoculation Single colonies of bacteria harbouring biobricks BBa_R0010 and BBa_K516032 were transferred to 5 mL of Lysogeny broth (LB) medium in 4 autoclaved tubes. These tubes were incubated at 37°C and shaken at 220 rpm overnight. LB contains 1 w/v% tryptone, 0.5w/v% yeast extract and 1w/v% NaCl. For LB agar plates, 1.5% bacteriological agar was used. DNA Extraction Plasmid extraction was done according to the MiniPlus DNA Extraction Kit protocol (“Mini Plus”, 2005). After the extraction, plasmid was tested with the Nanodrop machine, with the DNA concentration, 260/280 and 260/320 figures marked down. Digestion Digestion was conducted according to the modified protocol from NEB (“Digestion Protocol”, n.d.). For the restriction fragments preparation, the target fragments were digested with Xbal, Spel-HF and Pstl-HF provided by NEB. The total volume of each mixture is 35 μL. After that, incubate the tubes for 1 hour. The second digestion involved 2 sets of samples, one of them is the negative control group. After both digestions, gel electrophoresis was used to visualize the DNA bands. 1kb plus ladder and Lambda DNA/EcoRI+HindIII Marker were used as a reference for better observance. 0.8% and 1% of agarose gel were used with respect to the first and second digestion. After running the gel for 20 to 25 minutes, the result was observed through gel doc system. Gel purification Carry out gel electrophoresis using 1% agarose gel and 7μL columns. The procedures were then conducted according to protocol from Favorgen (“GEL/PCR Purification”, n.d.). After gel purification, the samples were tested with the Nanodrop machine for the DNA concentration, 260/280 and 260/320 figures. Ligation Ligation was carried out by following the protocol from NEB (“Ligation Protocol”, n.d.). Result and Interpretation Figure 1. Digested pSB1C3-BBa_R0010-BBa_K516032 candidates. 200 ng of DNA were digested by 1 unit of NEB PvuII-HF at 37°C for 2 hours. The result was analyzed on a 1% agarose gel pre-stained with 1x Midori Green. ED, JE, DA and NA are replicates prepared by Edward, Jenny, Daniel and Natalie respectively. Numbers indicate the identities of candidates. 0.5 μg of 1Kb Plus DNA Ladder (1kb) and 0.5 μg Lambda DNA/EcoRI+HindIII Marker (λ) were used as size marker. Figure 2. Red fluorescence image of pSB1C3-BBa_R0010-BBa_K516032 candidate colonies. Transformants of ligation product of pSB1C3-BBa_R0010-BBa_K516032 were observed under fluorescence macroscope for signs of mRFP. Transformants were first incubated at 37°C for 24 hours, followed by storage at 4°C for 20 hours before observation. Bright field (BF) images were taken with 50 ms exposure and gain 1 acquisition while 500 ms exposure and gain 2 acquisition were used for red fluorescence (RFP) images. D, E, J and N are replicates prepared by Daniel, Edward, Jenny and Natalie repectively. Scale bar = 5 mm. Discussion Conclusion Reference