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Latest revision as of 03:48, 28 September 2013

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Background

Regulated gene expression is one of the primary means by which phenotypic differences are generated among genetically identical cells (e.g., a human endothelial cell versus a human nerve cell). Nonetheless, the concept of inducible gene expression is often difficult for students (especially at the middle and high school level) to understand. This may be due, at least in part, to the lack of teaching tools designed to help students develop a conceptual framework upon which to build their understanding of this phenomenon. Therefore, in order to help students gain a better understanding of inducible gene expression, we developed "BactoArt", an multicomponent, inducible expression system that allows students to visualize gene regulation in action.

By placing different fluorescent protein (FP) color variants (e.g., cyan (CFP), green (GFP) or red FP(RFP)) under the control of specific inducible promoter, BactoArt enables students to "see" the effects of inducible gene expression where the corresponding inducers are applied. The specific color variant that is expressed is determined by the local environment of the bacterium. For instance, addition of the sugar xylose would induce the expression of an enhanced green FP (EGFP) variant under the control of the Xyl promoter (pXyl) while the addition of isopropylthiogalactoside (IPTG) would induce the expression of an enhanced cyan FP (ECFP) under the control of the Lac promoter (pLac). By patterning the inducers on a petri dish—using either a paintbrush or an inkjet printer--and then plating BactoArt bacteria, students are able to selectively induce the expression of a given FP. We believe that such hands-on activities will spark questions, leading to "teachable moments" pertaining to transcriptional regulation and the central dogma.

Strategy/Approach

To create the BactoArt plasmid (pBactoArt), inducible promoters and FPs (along with appropriate ribosome binding sequences (RBSs) and transcriptional terminators) were selected from the iGEM Standard Parts Registry and assembled using the "3A Assembly" protocol. Specifically, sub-parts consisted of 1) EGFP under the control of pXyl, 2) ECFP under the control of pLac and 3) EYFP under the control of the Hyc promoter (pHyc). Once constructed, the sub-parts are then assembled into a single plasmid, termed pBactoArt, encoding all three inducible expression modules and transformed into JM109 E. coli for expression (the JM109 strain of E. coli was chosen because the K strain of E. coli, from which dh5[alpha] are derived, encodes a dysfunctional lac repressor). The inducers used in these experiments are: 1) Xylose (to induce the expression of EGFP under the control of pXyl), 2) IPTG (to induce the expression of ECFP under the control of pLac) and 3) Formate/molybdate (to induce the expression of EYFP under the control of pHyc). To create BactoArt, inducers are first applied to a LB/agar plate containing the appropriate antibiotic and allowed to dry. Bacteria transformed with pBactoArt are then plated and incubated overnight at 37 C. The following day, fluorescence is induced by wide-field illumination.

Future Goals

In the future, we plan to incorporate additional promoters and FPs, some of which exhibit unique photophysical properties (e.g., the photo-switchable FP, Dronpa). This will expand the types of art that can be created using BactoArt (e.g., simple animation) and create exciting learning opportunities for students. Indeed, we envision that Bacto-Art can be a valuable tool to introduce middle and high school students to the concepts of inducible promoters and regulated gene expression. Moreover, the multi-component, multi-color expression system used by BactoArt may be useful in exploring the evolutionary stability of various promoter systems.