Research ArticleSYNTHETIC BIOLOGY

BioBits™ Bright: A fluorescent synthetic biology education kit

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Science Advances  01 Aug 2018:
Vol. 4, no. 8, eaat5107
DOI: 10.1126/sciadv.aat5107
  • Fig. 1 BioBits™ Bright: A portable, cell-free synthesized fluorescent protein library for teaching the central dogma of molecular biology and synthetic biology.

    (A) We describe here the development of an educational kit containing two laboratory modules using FD-CF reactions and a library of in vitro–synthesized fluorescent proteins. (B) In module I, students investigate how biological systems can be engineered by adding varying amounts of DNA template to FD-CF reactions. Titrating the amount of DNA template results in varying levels of fluorescent protein production, which are visible to the naked eye and under a blue or black light. (C) In module II, users design their own in vitro program using DNA encoding the fluorescent protein library and any of the DNA template concentrations investigated in module I. This module offers the opportunity to go through a user-directed design-build-test (DBT) cycle. All reagents used in these activities (freeze-dried reactions and plasmids) can be stored and transported without refrigeration, making them highly portable for use outside of the laboratory.

  • Fig. 2 High-yielding cell-free production of fluorescent protein library enables development of BioBits™ Bright.

    A 13-member fluorescent protein library was designed to include red, orange, yellow, green, cyan, and blue fluorescent protein variants and cloned into the cell-free expression vector pJL1. (A) Following CFPS for 20 hours at 30°C, soluble yields of the fluorescent protein library were measured via 14C-leucine incorporation. Values represent averages, and error bars represent SDs of n ≥ 3 biological replicates. (B) Soluble fractions were analyzed by SDS-PAGE and 14C autoradiogram. All library members expressed with exclusively full-length products observable by autoradiogram. (C) Images of FD-CF reactions expressing the fluorescent protein library under white light (top) and blue light (bottom).

  • Fig. 3 Controllable in vitro expression of diverse fluorescent proteins.

    FD-CF reactions were rehydrated with 25, 10, 5, 2.5, or 0 ng of template DNA encoding mCherry, mRFP1, dTomato, mOrange, or YPet and run for 20 hours at 30°C. (A) Results from experiments run by graduate students (experts), high school students, or middle and high school teachers are shown. In all cases, we observed a concomitant decrease in protein synthesis as the amount of DNA template was decreased. Values represent averages, and error bars represent average errors of n ≥ 2 biological replicates. (B) The variation in protein expression was marked enough to be observed qualitatively with the naked eye under both white light and blue light. Images are representative examples of experiments prepared by high school students.

  • Fig. 4 Design and execution of in vitro programs.

    Participants were asked to design, build, and test their own in vitro program with DNA in a 96-well PCR plate. Designs could include the mCherry, mRFP1, dTomato, mOrange, YPet, or sfGFP plasmids at concentrations between 0 and 25 ng (same template concentrations tested in module I), denoted with corresponding colors and opacity in the pictured designs (legend, bottom left). Successful designs included (A) a rainbow, (B) a periodic table, (C) a wildkit (the Evanston Township High School mascot), and (D) a game of Connect Four®. These biological programs were designed, built, and tested by untrained operators, demonstrating the potential of this laboratory for use in a classroom setting.

  • Fig. 5 Portable, low-cost equipment for teaching outside of the laboratory.

    (A) The eight-well imager is handheld and battery-operated for easy use (top) and can be used to image the six-member fluorescent library (bottom). We show FD-CF reactions expressing, from left to right, mCherry, mRFP1, dTomato, mOrange, YPet, and sfGFP. (B) The 96-well imager is also battery-powered and has a removable lid for easy use (left). In vitro biological programs can be imaged using our custom 96-well imager with similar performance as a laboratory imager (right). (C) The portable incubator accommodates up to 96 standard PCR tubes and has a removable, insulating lid for maintaining reaction temperature at its two set points, 30° and 37°C (left). Fluorescent protein yields using our incubator set at 30°C are at least 50% of those achieved using a laboratory incubator (top right) and produce fluorescence that is visible in our handheld eight-well imager (bottom right). Values represent averages, and error bars represent average errors of n = 2 biological replicates.

  • Table 1 Diverse fluorescent protein library enables educational kit development.

    A 13-member fluorescent protein library was designed to include red, orange, yellow, green, teal, and blue fluorescent protein variants, which were cloned into the in vitro expression vector pJL1. PDB accession numbers are provided if the protein (or a closely related variant) has been crystallized.

    ProteinColorExcitation
    (nm)
    Emission
    (nm)
    PDB entry
    mCherryRed5876102H5Q
    mRFP1Red5846072VAD
    eforRedRed5876102VAD
    dTomatoOrange554581
    mOrangeOrange5485622H5O
    YPetYellow5175301F0B
    sfGFPGreen4855282B3P
    mTFP1Cyan4624924Q9W
    CyPetCyan4354773I19
    AquamarineCyan4204742WSN
    mTagBFP2Blue3994543M24
    mKalama1Blue3854564ORN
    eBFP2Blue3834481BFP

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/8/eaat5107/DC1

    Fig. S1. Diversity of the fluorescent protein library facilitates evolution curriculum.

    Fig. S2. Fluorescent protein library expresses with soluble, full-length products observed by SDS-PAGE and autoradiogram.

    Fig. S3. FD-CF reactions tolerate a range of incubation temperatures.

    Fig. S4. DNA template is not limiting for in vitro sfGFP synthesis due to relatively high initial rates of protein synthesis.

    Fig. S5. Orange and yellow filters enable imaging of diverse fluorescent proteins in portable imagers.

    Fig. S6. FD-CF reactions can be run in a laboratory-free environment using low-cost, portable imagers and incubators.

    Fig. S7. Standard curves for converting fluorescence to protein concentrations.

    Table S1. Cost analysis of portable imagers and incubators.

    Table S2. Cost analysis for BioBits™ Bright.

    Table S3. Cost analysis of FD-CF reactions.

    Table S4. Plasmids used in this study.

    Curriculum S1. Let it glow!

    Curriculum S2. What factors affect CFPS yields?

    Curriculum S3. Synthetic biology: Looking to nature to engineer new designs.

    Curriculum S4. How fast is it really?

    Curriculum S5. Super power protein!

    Data S1. This file contains example student-generated fluorescence data from the tunable protein expression laboratory activity (Fig. 3) and includes time-course data for modeling protein synthesis as an enzymatic reaction with varying amounts of substrate (DNA template).

    Folder S1. This folder contains FreeCAD files and circuit diagrams to enable user construction of portable imagers and incubators.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Diversity of the fluorescent protein library facilitates evolution curriculum.
    • Fig. S2. Fluorescent protein library expresses with soluble, full-length products observed by SDS-PAGE and autoradiogram.
    • Fig. S3. FD-CF reactions tolerate a range of incubation temperatures.
    • Fig. S4. DNA template is not limiting for in vitro sfGFP synthesis due to relatively high initial rates of protein synthesis.
    • Fig. S5. Orange and yellow filters enable imaging of diverse fluorescent proteins in portable imagers.
    • Fig. S6. FD-CF reactions can be run in a laboratory-free environment using low-cost, portable imagers and incubators.
    • Fig. S7. Standard curves for converting fluorescence to protein concentrations.
    • Table S1. Cost analysis of portable imagers and incubators.
    • Table S2. Cost analysis for BioBits™ Bright.
    • Table S3. Cost analysis of FD-CF reactions.
    • Table S4. Plasmids used in this study.
    • Legends for curricula S1 to S5
    • Legend for data S1
    • Legend for folder S1

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Curriculum S1 (Microsoft Word format). Let it glow!
    • Curriculum S2 (Microsoft Word format). What factors affect CFPS yields?
    • Curriculum S3 (Microsoft Word format). Synthetic biology: Looking to nature to engineer new designs.
    • Curriculum S4 (Microsoft Word format). How fast is it really?
    • Curriculum S5 (Microsoft Word format). Super power protein!
    • Data S1 (Microsoft Excel format). This file contains example student-generated fluorescence data from the tunable protein expression laboratory activity (Fig. 3) and includes time-course data for modeling protein synthesis as an enzymatic reaction with varying amounts of substrate (DNA template).
    • Folder S1 (.zip format). This folder contains FreeCAD files and circuit diagrams to enable user construction of portable imagers and incubators.

    Files in this Data Supplement:

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