Part:BBa_J119044:Experience

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Applications of BBa_J119044

Users should not use the version of this part if cloned into pSB1A8 (Part:BBa_J119043). We have found that J119044 will recombine with the two transcriptional terminators (TTs) built into pSB1A8. Therefore, we recommend that people use the version cloned into pSB1A2 with BsaI site removed from the ampicillin resistance gene. pSB1A2 normally has a BsaI site in the plasmid, but we have engineered a version of pSB1A2 with the BsaI site removed.

Students learn better through hands-on laboratory experiments if they are genuine research. We wanted to enable introductory biology students to conduct real research in synthetic biology to discover promoters that could be easily regulated. Golden Gate Assembly (GGA) makes cloning and testing new plasmids easy enough to fit into traditional laboratory periods because users do not need pre-digested plasmids or gel-purified inserts. We investigated how to do GGA with the least amount of hands-on time possible. A range of experimental conditions produce excellent results (see table 1), so this approach could be used on campuses around the world. With GGA, Bio113 students can do research by testing the function of new promoters.

With GGA, students can clone promoters into plasmids by using ligation solutions containing both BsaI and ligase. These solutions go through different amounts of temperature cycles, and different methods are used to make the promoters (such as oligo assembly, PCR amplification, or just using the entire plasmid containing the promoter). Several combinations of cycles and methods produced positive results, meaning that plated cell colonies show red fluorescent protein (see figures 2 and 3). The right combination depends on how much time and what resources the professor has (see table 1).

Figure 1: Ways to build the promoter




Figure 2: 1 µL raw PCR product, 10 cycles, trace amount red, circles indicate a few red colonies




Figure 3: Spin column-cleaned oligos, 20 cycles, about 30% red




Figure 3: Cleaned PCR product, 10 cycles, about 50% red




Figure 4: Cleaned PCR product, 30 cycles, mostly red




Table 1: Combinations of GGA that work

User Reviews

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