RBS (BBa_B0032), mRFP CDS (BBa_E1010) and double terminator (BBa_B0015)
Standard Assembly
[[Image:|thumb|left|700px|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.]]
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.
