Notebook – Results – 2. Confirmation of DNA sequence

As shown in Figure 1 (2AGL_DT), we confirmed the construction of crr gene encoding IIA(Glc) from E. coli into Biobrick format – flanking by restriction sites of EcoRI and XbaI at the prefix, and SpeI and PstI at the suffix – followed by the sequence of double terminator (BBa_B0015). The other constructs which able to be confirmed by DNA sequencing was the parental plasmid containing crr gene only. While the rest of plasmids did not contain expected sequence.

While the final construct to be expected, namely BBa_J04450 followed by constitutive promoter/RBS/crr/double terminator was actually the sequence of BBa_J04450 only as shown in figure below. The data from forward sequence and reverse sequence (Figure 2; J04450_CP_RB_2AGL_DT FWD/REV) revealed the prefix and suffix sequences clearly.

However, we can confirm the sequence of basic part in our project namely BBa_J23119 (constitutive promoter), BBa_B0034 (RBS) and BBa_B0015 (double terminator) alone. Thus for future works, we can confirm to use the plasmid for construction.

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