pClone: Synthetic Biology Tool Makes Promoter Research Accessible to Beginning Biology Students

Abstract

The synthetic biology lab module pClone is ideal for beginning students to conduct authentic research—inexpensive and easy to prep. Students clone and characterize promoters and share their results in research-grade databases with significant learning in the core concept of information and several core competencies described in Vision and Change.

Implementation of a Collaborative Series of Classroom-Based Undergraduate Research Experiences Spanning Chemical Biology, Biochemistry, and Neurobiology

“…Thus, we think that the measured pre- to postcourse gains were due to student learning and development of the context-specific experimental design skills that were reinforced through their experiments and writing assignments within our CUREs. Overall, the gains in experimental design mirrored the gains in experimental logic reported for a recently modified SEA-PHAGES course-based research experience (Staub et al ., 2016) and the content-based gains observed for a variety of other research courses with a range of levels and topics (Shaffer et al ., 2010; Campbell et al ., 2014; Harvey et al ., 2014; Russell et al ., 2015). Similar increases in both lower- and higher-order cognitive skills were recently reported for students participating in both CUREs and in independent AREs; interestingly, the pre- to postcourse increases were greater for CURE students, who, on average, began at a lower achievement levels than students in AREs, suggesting the importance of CUREs in narrowing student achievement gaps (Shapiro et al ., 2015).…”

rClone: A Synthetic Biology Tool That Enables the Research of Bacterial Translation

“…rClone uses Golden Gate Assembly (GGA), which eliminates the need for gel purification of DNA and enables the standardization of assembly for molecular cloning (Weber et al, 2011). We leveraged the simplicity and reliability of GGA for rClone, which enables the cloning of RBSs, as we did previously for pClone, which enables the cloning of promoters (Campbell et al, 2014). After RBSs and promoters are cloned, rClone and pClone to enable the measurement of their func- …”

“…Each tool leverages the ease with which GGA can be used to clone user-defined regulatory elements and measure their effects on expression of a reporter gene. pClone Red (Bba_J119137) and pClone Blue (Bba_J119313) enable students to conduct promoter experiments of their own design Campbell et al, 2014;Eckdahl & Eckdahl, 2016;. tClone Red (Bba_J119367) facilitates investigation of transcriptional terminators that use alternative RNA folding states to control gene expression.…”

Mutational Analysis of Transcriptional Initiation in Bacteria

“…The sequence of the -35 region is identical to the -35 region consensus of TTGACA. The method by which we introduced mutations into Ptac involves a system recently developed by our research group (Campbell et al, 2014). Figure 2 illustrates the structure of our pClone Red plasmid for cloning and measuring bacterial promoters.…”

“…The annealed oligonucleotides were diluted with water to the same concentration as the pClone Red destination vector (Campbell et al, 2014; http://parts.igem.org/Part:BBa_J119137 in the Registry of Standard Biological Parts) to provide a 1:1 molar ratio of promoter insert to pClone vector in the GGA reaction. For Mutant promoters in the form of annealed promoter oligonucleotides can be cloned into pClone Red with Golden Gate Assembly (GGA) using BsaI and DNA ligase.…”