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 Table 1. BioBricks used in this investigation. 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. 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. 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. 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 Electrophoresis 0.8% and 1% of agarose gel were used with respect to the fragments isolation and diagnostic digestion check. 1kb plus ladder and Lambda DNA/EcoRI+HindIII Marker were used as a size marker. After running the gel for 20 to 25 minutes, the result was documented through gel doc system. Gel purification The protocol was adopted from Favorgen. 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. The volume of reaction was changed to 10 μL. Result and Interpretation Figure 2. 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. Based on past experiences of assembling the commonly used promoter BBa_R0010, colonies with bright red color were expected to be observed after being cultivated overnight inside the incubator. From Figure 2, there are no apparent red colonies observed after 24 hours incubation. However, red pigments were only observed form the pellet obtained after centrifugation of cell culture. After 40 hours, there were still no apparent red colonies observed from the plate but some were observed under fluorescent macroscope, as shown in Figure 3 below. Figure 3. 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. The red colonies were further selected and screened by the diagnostic digestion check. From the database of the Benchling, Pvull-HF was selected as restriction enzyme and the expected gel electrophoresis results generated by Benchling is shown in Figure 4 ,while the experimental results of the restriction digestion check is shown in Figure 5 below. Figure 4. Expected results of diagnostic restriction generated by Benchling. Figure 5. 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. Discussion Based on the past experiences of assembling the commonly used promoter BBa_R0010, colonies with bright red color were expected to be observed after being cultivated overnight inside the incubator. However, red pigments were observed after centrifugation, in some samples containing the colonies picked after 24 hours . After 40 hours, there were no apparent red colonies observed from the plate but some were observed under fluorescent microscope. Since the experimental result deviates from our expectation, there are few possible reasons for the weak fluorescent signal detected. First and foremost, the yield of the RFP could be low, due to the incorrect sequence in the regulatory elements such as the promoters, Ribosome Binding Site(RBS) and even the Origin of Replication (ORI), because they control the transcription rate, translation rate, and the amount of plasmid copies in the bacterial respectively, which would affect the expression of the operons. Another reason could be incorrect protein sequence. Since the sequence would affect the conformation of the protein, if the sequence is different from what we expected, some of the characteristics of the protein, such as the maturation time, half-life could be different, resulting weak or delayed fluorescent signal detected. Conclusion To conclude, mRFP producing E.coli was successfully created. Since the fluorescent signal was delayed and weaker than expected, it is suggested that further DNA sequencing should be done in order to confirm the sequence is consistent with the database, in order to find out the reason for the unexpected results. Reference Transformation Protocol. (n.d.). Retrieved January 28, 2016, from https://www.neb.com/protocols/2012/05/21/transformation-protocol Mini Plus Plasmid DNA Extraction System User’s Guide [PDF]. (2005). Taiwan: Viogene-BioTek Corporation. Digestion Protocol for BioBrick Assembly Kit (E0546). (n.d.). Retrieved January 28, 2016, from https://www.neb.com/protocols/1/01/01/digestion-protocol-e0546 Ligation Protocol with T4 DNA Ligase (M0202). (n.d.). Retrieved January 28, 2016, from https://www.neb.com/protocols/1/01/01/dna-ligation-with-t4-dna-ligase-m0202