Team:William and Mary/Experiments
Experiment
Protocols
We have uploaded all of our protocols for lab tasks as a zip file.
A list of contents is found here:
- 3G Assembly (contains instructions for Golden Gate and PCR amplification of Golden Gate product. Additional protocols must be used for subsequent gel extraction of PCR product and Gibson of transcriptional units into vectors)
- Annealing 3G Adapters (how to anneal UNS sequences when ordered as single-stranded oligos)
- Biofilm Lithography (follows protocol laid out in 2018 PNAS paper by Jin & Riedel-Kruse)
- Centrifuge (how to properly balance and operate the centrifuge)
- Colony PCR (assess colonies for successful transformation)
- DpnI Digestion (remove methylated template DNA after PCR)
- Electroporation of M. smegmatis (for competent M. smegmatis cells)
- Filter Sterilization
- Gel Electrophoresis
- Gel Extraction (follows 3G Assembly Protocol. Also aids in isolation of desired DNA when multiple bands are present on gel electrophoresis gels)
- Gene Block Resuspension
- Gibson Assembly (final step for plasmid assembly via 3G DNA Assembly. Combines transcriptional units into a backbone utilizing UNS overlaps. Gibson Assembly is also used to place parts with “Pad” adapter sequences into William and Mary’s “Pad 1C3,” a cloning backbone)
- Inoculation for Miniprep
- Making Competent Cells (makes JS006 experimental cells competent. Competent DH5-alpha cells are bought from NEB)
- Making LB Agar Plates
- Midiprep
- Miniprep
- Nanodrop (proper operation and cleaning of Nanodrop used to quantify DNA concentration and purity)
- PCR Purification
- Plate Reader (how to properly operate and collect data using our Synergy plate reader)
- Resuspension of primers (received in dried form)
- Standard PCR
- Transformation
Methods
Induction and Assessment of Adhesins
To assess pDawn-AG43, we strictly followed the methods of "Biofilm Lithography enables high-resolution cell patterning via optogenetic adhesin expression" and the related Jove information video (Jin & Riddle-Kruse, 2018). Though we used a Magnasonic LED Pico Projector to project blue light, rather than the original Ivation Pro4 projector used by Jin and Riedel-Kruse, we followed other instructions regarding inoculations, dilutions, and blue slide preparation on Microsoft Powerpoint. These methods were summarized in a protocol we drafted called “Biofilm Lithography.” Blue-light induction of both pBlind-curli containing circuits followed this protocol, as well. This protocol, which provides instructions for crystal violet staining of AG43, is contained within the zip file.
To assess amyloid fiber production, we utilized the Congo Red spin-down assay designed by Harvard iGEM’s 2017 team, OptiPoly. 3 mL LB broth was inoculated with 3 uL colony solution (one colony in 10 uL nuclease-free water) and the culture was allowed to grow overnight. The next day, cells were stained via a 0.015% Congo Red solution, then centrifuged and pelleted. If cells had expressed curli fibers, the Congo Red stain would bind the fibers and end up in the cell pellet, resulting in a lighter supernatant than samples lacking curli fibers. The color of the supernatant, along with the OD600 of the original culture sample, was quantified using a plate reader. However, difference in pellet color was often apparent to the naked eye. As well, aggregation in the bottom of inoculated glass tubes was often observed prior to Congo Red staining. See the full, step-by-step protocol here.
Visible differences prior to quantitative data collection: visible aggregation on the bottom of glass culture tubes with induced samples (left photo, right tube). Uninduced samples show no such aggregation (left photo, left tube). Before plate reader analysis, Congo Red results are visible (right photo). The induced sample (rightmost 1.7 mL centrifuge tube) has a redder pellet than the uninduced sample (middle 1.7 mL centrifuge tube), which closely resembles the untransformed JS006 negative control (leftmost 1.7 mL centrifuge tube).
To assess inducible circuits, two cultures were inoculated per colony solution, ensuring that the cells within both tubes were genetically identical. One culture was induced while the other was not. To assess constitutive circuits such as J23107-curli, equal numbers of experimental and negative control (untransformed JS006) tubes were inoculated to enable paired t-tests.
IPTG induction for curli fibers was eventually accomplished using 10 uL 1M IPTG/3 mL, ~3.3 mM final concentration (see Results page for results of preliminary experiments with various inducer concentrations). IPTG induction of SaSuhB was accomplished with final IPTG concentration of 10 mM, while IPTG induction of fap was accomplished with a final concentration of ~24.4mM. Untransformed JS006 were inoculated as negative controls for IPTG-inducible curli experiments. pBbB8K-csgBACEFG, a verified synthetic curli operon from Dr. Neel S. Joshi at Harvard's Wyss Institute, served as a "positive control" to exemplify what robust curli expression should look like. See the results page for more information.
Quorum Sensing and Distance-Dependent Patterning
To assess our quorum sensing circuits’ ability to establish distance-dependent patterning, we devised a “ring experiment” utilizing the blue light-induced pDawn system. First, we created stocks of competent JS006 cells with the pDawn-AG43 plasmid. Then, we cotransformed our quorum sensing circuits into these competent cells, so that both sender and receiver strains had the pDawn-AG43 plasmid. Since our cells were cotransformed with this plasmid, we could use blue light biofilm lithography to place a small circle of sender strain cells on a petri dish. The next day, we aspirated the liquid media (leaving the sender strain biofilm on the plate) and replaced it with M63 inoculated with receiver strain bacteria. We again used biofilm lithography to create a biofilm, this time locating receiver strain bacteria in a ring surrounding the circular sender strain biofilm. After overnight placement of this ring biofilm, we induced the sender strain bacteria using a thin lawn of M9 + IPTG (1.5 mL total volume with 10 mM final concentration of IPTG; 15 uL 1M IPTG and 1450 uL M9). We waited two hours, allowing time for induced sender strain bacteria to produce HSL, for this HSL to diffuse outward into the ring of receiver strain bacteria, and for the ring of receiver strain bacteria to respond to the HSL and produce mScarlet. Results were visualized using a dissection scope.
Theoretically, the diffusion of HSL from the sender strain results in distance-dependent patterning. Fluorescence should be strongest in a ring surrounding the circular sender biofilm, and this ring of fluorescence should fade outwards in a gradient (since HSL, the inducer of this fluorescence, decreases in concentration as it diffuses away from the sender circle). Though presence of both sender and receiver strains was confirmed, and induction of the receiver ring by the sender was visualized, the expected gradient of fluorescence was not observed (see results page). The entire outer ring appeared equally fluorescent, which we attribute to over-induction of the sender strain by IPTG (and thus over induction of the receiver strain by HSL). Following this experiment, we realized that our biofilm was larger in size than that simulated by the math modelling team. We asked them to expand the scale of their model, as well as model different IPTG concentrations and incubation times. Future experiments will follow their suggestions for ideal induction of the sender strain.
Though our final ring experiment proved inconclusive, “benchmark” plate reader experiments with prototype circuits allowed us to assess the functionality of our quorum sensing circuits. For example, plate reader data from prototype circuits confirmed that 1) sender strain bacteria are successfully induced by IPTG 2) receiver strain bacteria are successfully induced by HSL 3) sender strain bacteria can successfully induce receiver strain bacteria when induced by IPTG. To verify conclusion 1, colonies of a prototype sender circuit (IPTG induction results in mScarlet production) were exposed to IPTG and red fluorescence was monitored over time. To verify conclusion 2, receiver strain were directly induced with HSL and fluorescence was monitored over time. To verify conclusion 3, both sender and receiver cells were grown together in one well. Fluorescence was measured over time after addition of IPTG. Fluorescence in the third scenario was further visualized by microscopy.
As well, prototype and final quorum-sensing circuits were sequence confirmed by Sanger sequencing on both strands by Epoch Life Sciences.