BACTO-ART A Multicomponent Inducible Expression System for Teaching and Discovery

1. BACTO-ART: A Multicomponent Inducible Expression System for Teaching and Discovery Derrick Azorlibu1,Ebony Stadler2, ShereseMann1, Maleek Richardson3, SarinaVeale2,, YannickTuwamo1, MeshaGuinyard1,4, Kenyon Jones1, KalynnHosea1, D. Alex Page1, Kelsie M. Bernot1and Robert H. Newman1 Departments of 1Biology, 2Biomedical Engineering, 3Applied Mathematics, and 4Chemistry, North Carolina A&T State University, Greensboro, NC 27411 Construction of a Two Color System: 3A Assembly Inducible Expression of FP Color Variants: SDS-PAGE Analysis EcoRI XbaI Scar EcoRI XbaI SpeI PstI EcoRI XbaI SpeI PstI 2hrs 6hrs 24hrs - - - RFP pLac Inducer: I X I X I X XylR pXyl RFP pLac EGFP SpeI PstI EcoRI XbaI BACTO-ART A Multicomponent Inducible Expression System for Teaching and Discovery pXyl- EGFP pLac- RFP MW + + 170 Part 2 130 Part 1 Chlr Chlr 100 Ampr 70 55 SpeI PstI 40 35 25 XylR pXyl EGFP 15 Parts Key 10 : promoter Results after 24 hours of incubation : RBS : FP variant Composite Part : Term Ampr • Figure 3. Inducible Expression of FP color variants as visualized by Coomassie-stained SDS-PAGE gel. Whole cell bacterial cell lysates were prepared in SDS-PAGE loading buffer after the indicated induction period. GFP and RFP run at an apparent MW of 27-30kD. • Figure 1. Constructionof2 color inducible expression system using 3A Assembly. Two single color expression systems, each of which is composed of a distinct fluorescent protein color variant under the control of a differentinducible promoter, were isolated from the iGEM Registry of Standard Biological Parts (BioBrick Collection).The first operon (Part 1) included, from 5’ to 3’: the lactose promoter (pLac), a ribosome binding site (RBS), red fluorescent protein (RFP)and a tandem transcriptional termination sequence (Term).The second operon (Part 2) included the the xylose promoter (pXyl), a RBS, enhanced green fluorescent protein (EGFP), and a terminator sequence. Introduction Applications: Bacto-Art as a Teaching Tool 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, Bacto-Art 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 RFP 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" about transcriptional regulation and the central dogma of biology. Inducible Expression of FP Color Variants: Fluorescence Timecourse Future Directions Continue developing the project so that the E. coli strain used can express, at a minimum, four different colors. Other Fluorescent Proteins will include Cyan FP (CFP), Far-Red FPs (FRFP), etc. Project Goals • Create a system that can make fine detailed art pictures. • Using an inkjet printer, put sugars in a cartridge to print out the desired picture. UV light • To create a multicomponent, inducible expression system that can be used to illustrate fundamental biological concepts, such as gene regulation and the central dogma of biology, to middle and high school students. • To gain hands-on experience in the fields of molecular and synthetic biology • To expand NC A&T’s international Genetically Engineered Machines (iGEM) synthetic biology team • Create photoswitchable FP Bacto-Art • Moving Pictures: The proteins react to different wavelengths of light to glow at a certain time. • Detection systemto assess food & water purity • To trace contamination in canned foods or water sources before consuming and distribution blue light Acknowledgements • Figure 2. Inducible Expression of FP color variants by measuring relative emission for GFP (425nm excitation / 485nm emission) and RFP (613nm excitation / 675nm emission) using a fluorescence spectrophotometer (graphs) or by naked eye using normal indoor fluorescent lighting (photograph of bacterial cultures). For graphs, expression is normalized to the no induction control for each time point. We would like to thank all of the “Greensboro-Aggies” iGEM team members. This work was funded by an educational grant from NSF-BEACON (DBI-0939454) and the NSF I3 grant (DBI-1038160).