## 3 Results
At 6 days, the particles in the ATCC31749 culture were mostly larger than 20 \(\upmu\)m in diameter. However, the particles from the _crdS_ disruptant did not increase in diameter. This result supported the hypothesis that curdlan production was indeed associated with floc formation, and that the loss of curdlan production is associated with failure to form high numbers of flocs in liquid cultures. 96-well plate assay reveals onset of curdlan production. How well does expression of the _crdASC_ genes correlate with curdlan production? A tight correlation would provide more support for the idea that curdlan production is controlled by _agrobacteriumium_ at the level of _crdASC_ gene expression. On the other hand, a poor correlation would indicate that curdlan production was controlled by additional unknown factors. A previous study using transcription analysis found a 100-fold upregulation of the _crdASC_ genes upon the transition to nitrogen starvation [37], which lends weight to a tight correlation. To recapture this result, as well as to have a more sensitive, rapid and cheaper technique for the detection of both gene expression and curdlan production, a novel assay capable of a high throughput was developed. Until now, detection of curdlan production had been performed qualitatively via growth on agar containing aniline blue (where curdlan producing colonies turn intense blue while non-producing colonies remain white) or quantitatively following the extraction of curdlan via alkaline solution, neutralization, washing, drying and weighing. The extraction method is relatively laborious and impractical for analyzing a high number of samples, while growth on standard aniline blue agar plates is impractical for obtaining temporal data. For developing this assay, I took advantage of the knowledge that curdlan accumulation in bacterial cultures is easily detectable using the (1\(\rightarrow\)3)-\(\beta\)-glucan-specific dye aniline blue [27]. For the setup, 100 \(\upmu\)L of 1% agar containing aniline blue and nutrients from ADB (_Agrobacteriumium_ define broth) was placed in each well in a 96-well plate. The rational for using agar in the 96-well plate was to minimize the volume of liquid cultures. This reduces the problem of measuring growth in liquid cultures with strong floc formation and the associated variation between measurements. Once the agar was set, 20 \(\upmu\)L of ADB inoculated with _agrobacteriumium_ was pipetted onto the agar, and the plate was covered with a transparent plastic lid to prevent drying of the agar and incubated in a Tecan reader at 30 \({}^{\circ}\)C up to 72 h. The Tecan reader was set to measure the cultures growing on the surface of the agar in the well automatically at 3 h intervals. For the 96-well plate assay, reporter strains were prepared (using C58 and ATCC31749) in which the gene for mVenus was fused (translational fusion) to _crdA_ (see Figure 1 for details of the genetic construct). Using these strains, I specifically looked for correlation between the onset of _crdA_ gene expression (detected as mVenus fluorescence) and the onset of curdlan production (as detected via aniline blue/curdlan fluorescence). Absorbance at 600 nm (providing an indication of cell density, Figure 3A) was strongly impacted by curdlan accumulation in strain ATCC31749 at around 30 h, since binding between aniline blue and curdlan results in an intense blue colour that also absorbs light at 600 nm (Figure 3A). The large error bars are likely because of the irregular colony surface that accompanies curdlan production. Growth of C58 was weaker than that of ATCC31749. Curdlan binds to aniline blue, and the complex emits red fluorescence at 548 nm when excited by light at 515 nm. Fluorescence from the aniline blue-curdlan complex was apparent in cultures of ATCC31749 after 30 h, but not in cultures of C58 (Figure 3B).