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 In recent decades, there are lots of researches regarding recombinant DNA techniques. This greatly facilitates the research in the field of synthetic biology. In this project, our aim is to use recombinant DNA technique to create bacteria which harbor recombinant plasmids with the gene encoding mRFP. We employed standard assembly method, which includes restriction enzyme digestion and ligation, to construct the recombinant DNA. Red colonies were originally expected to be shown on the plate after transformation. However, no conspicuously red colonies could be found with naked eyes after 2 days but could only be seen under fluorescence macroscope. Introduction Standard BioBricks Assembly allows different types of BioBricks that contain desired DNA fragments to be assembled by restriction digestion and ligation. The objective of this project is to investigate the assembly method by creating mRFP containing cell from the BioBricks BBa_R0010 and BBa_K516032. BBa_R0010 is a lac-inducible promoter while BBa_K516032 is an intermediate containing BBa_B0032 RBS, BBa_E1010 Red Fluorescent Protein and a BBa_B0015 double terminator. By combining the two BioBricks and transformed the recombinant plasmid into the cell, red fluorescent protein producing E. Coli could be obtained. The details of the BioBricks used are shown in Table 1. BioBricks used in this investigation. Methods and Materials BioBricks BioBricks Size (bp) plasmid remarks BBa_R0010 200 pSB1C3 Lac-inducible promoter Bba_K516032 862 pSB1C3 RBS (BBa_B0032), mRFP CDS (BBa_E1010) and double terminator (BBa_B0015) Standard Assembly Figure 1. Standard assembly of BBa_R0010 and BBa_K516032. As illustrated in Figure 1, pSB1C3-BBa_R0010 was cut by the restriction enzyme Spel-HF and Pstl-HF and serve as a vector in the ligation, while pSB1C3-BBa_K516032 was cut by Xbal and Pstl-HF and isolated as an insert. After the restriction digestion, the interested fragments were firstly purified through gel purification, and then mixed and ligated using T4 ligase. The end product of the ligation was transformed into the competent cell. Transformation Following the modified protocol provided by NEB (Transformation Protocol, n.d.). 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 instead. Inoculation Single colony of bacteria harbouring biobricks BBa_R0010 or BBa_K516032 were transferred to 5 mL of Lysogeny broth (LB) medium. The culture was 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, 1.5w/v% 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 protocol from NEB (“Digestion Protocol”, n.d.). For the restriction fragments preparation, the target fragments were digested with Xbal, Spel-HF and Pstl-HF purchased from NEB. For diagnostic digestion, the enzyme used was PvuII-HF purchased from NEB. Gel purification The protocol was adopted from Favorgen (“GEL/PCR Purification”, n.d.). DNA were extracted from a 0.7% agarose gel. The purified samples were tested with the Nanodrop machine for the DNA concentration, 260/280 and 260/320 figures. Ligation Ligation was carried out using the protocol from NEB (“Ligation Protocol”, n.d.). The volume of reaction was changed to 10 μL. Result and Interpretation Figure . Plates of pSB1C3-BBa_R0010-Bba_K516032 candidates colonies after 24 hours incubation. After ligation and transformation of the recombinant DNA, cells containing pSB1C3-BB1_R0010-BB1_K516032 plasmids were cultivated on LB agar plates with CHL and incubated at 37°C for 24 hours 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