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Team:iHKU/result
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ResultsCONTENTS:
Generation of cheZ knock-out strain by using λ-Red mediated deletion strategy We first constructed a plasmid (PBSKcl) that contained a chloramphenicol (Cm) resistance module as a selectable marker. These were used as templates for the PCR amplification of DNA ‘targeting cassettes’, to delete the CDS of interest. PCR primers each included 45–50 bases homologous to the chromosome immediately upstream (forward primer, sense strand) and downstream (reverse primer, antisense strand) of cheZ gene, as well as 17 bases homologous to the plasmid template.
Fig. 1 Diagram illustrating the strategy used to delete cheZ (A) and agarose gel electrophoresis of confirmation PCR products (B). B, 1-4, MA6 (cheZ replaced with cm); 5, iHKU101; 6, MG1655. Thereafter, we applied cells at log phase onto soft agar plate to test its motility. Cells lacking cheZ tumble incessantly and are essentially nonmotile (Fig2.B), while the parental strain, MG1655, migrated well (Fig2.A).
Fig.2 Migration of strains MG1655 and iHKU101.
Introduction of cheZ restored the motility of strain iHKU101
Fig.3 SDS-PAGE and western blotting analysis of CheZ expression (A) and migration of strains iHKU106 (B).
We confirmed motility is restorable (or inducible) in E. coli. We proceeded to investigate whether a unique pattern can be obtained by our density-dependent motility design. We constructed the 2 strains as described in the Design Section and let them, together with controls, to grow on soft-agar-plates following the “pattern development on soft-agar-plate” protocol. Specifically, the strains were pre-culture from overnight inoculums, when reach OD600 ~0.8, 2 μl of the culture was applied to the center of a soft-agar-plate and the plate was subsequently incubated overnight. We obtained quite different patterns amongst different strains. Circle pattern from MG1655 (wild type Control) In the case of the wild type, the obtained pattern was a circle (Fig. 4). As can be seen in the density profile, cells are uniformly distributed across the area covered. The diameter of the cell mass is roughly 7.5cm. Similar patterns were also observed in modeling reuslt (Fig. 4).
Fig.4 Pattern of MG1655.
Fuji-Mount pattern from iHKU105 But in the case of iHKU105 (design), a different pattern was observed compared to the wild type strain. In particular, the center of the circle is high in density (Fig. 5). The diameter of the cell mass is roughly 4 cm. Similarly, we got a pattern by our model, choosing an increasing function Dρ(h) (modeling).
Fig.5 Pattern of iHKU105. Ring-like pattern In our model, if the Dρ(h) decreased smoothly near the threshold, an amazing identical ring-like pattern was observed (modeling).  Fig.6 Pattern of iHKU102.
Effect of genetic circuit modification on pattern formation As predicted by modeling, if the graded change of cheZ expression over the cell density in iHKU102 could be tuned to bi-stable steady-state responses, the ring-like pattern formed by iHKU102 will be different, i.e. a multiple ring-like pattern will be obtained. Haseltine et al. demonstrated if the expression of LuxI, LuxR, and target proteins were all regulated by pluxI, a bi-stable switch would be achieved [2]. So in iHKU117, pluxRI2 was replaced with pluxRI3, by which luxR and luxI are controlled by positive auto-feed back. Interestingly, a different pattern really appeared. It’s apparent that the inner area shrank, and there’s a circle of cells with high density surrounding a ring of cavity. Outside this circle of cells, there’s the other weaker ring of cavity (Fig. 7).
Fig.7 Patterns of iHKU102 and iHKU117. Funny patterns 
Fig8. Funny pattern with two or more initial droplets in modelling(left)and in experiment(right) Importance of genetic circuits in pattern formation To determine the importance of introduced genetic circuit in this ring-like pattern, a series of control strains were generated (Plasmids and strains). As shown in Fig.9, either loss of luxR (iHKU104), luxI (iHKU103), CI (iHKU109), luxR and luxI (iHKU115), or CI and cheZ (iHKU118) resulted in the failure of development of ring-like pattern, suggesting that a complete genetic circuitry is required.
Fig. 9 photograph of control strains Characterization of factors controlling ring-like pattern formation We cultured the strains at room temperature (23oC), 32oC, and 37oC to investigate the effects of temperature on pattern development. Except for the time to form the ring-like pattern, the overall pattern appeared to be unaffected by temperature (data not shown). During our experiments, we sought to determine the effects of humidity of the environments on the pattern formation. When the humidity is too low, the water in the agar plate will be evaporated into the air rapidly, the agar concentration will rise, which will then cause the change of the diffusion coefficient of cells.
Fig.10 effects of water amount on humidity During our experiments, it is known that the agar concentration of the soft-agar plates is an influential factor to the development of the pattern. We investigated this factor by growing our strain on plates over a range of agar concentration. As shown in figure 11, the ring of void becomes clearer and clearer as agar concentration increases. This sheds light to the effects of migration rate of cells, since denser the agar, harder the cell migrate in it.
Fig.11 effects of agar concentration on ring-like pattern
iHKU101 is measured as a control for the experimental strain. After two times of precultures, the initial cell concentration was diluted to be OD600~0.05, and it entered exponential phase after two doublings and went on to the steady phase after 100 minutes with an OD600~1.6. The earlier appearance of the steady phase may be due to the limitation of nutrient or the accumulation of metabolic wastes.
Fig. 12 Growth curve of iHKU101 with the doubling time ~ 20.0 min The doubling time of iHKU102 is generally longer than iHKU101 (more than 30min) even without antibiotics (Fig.13). The initial OD is also ~0.05 and it enters exponential phase immediately in the experimental culture, and remain in the phase till 300 min.
Fig. 13 Growth curve of iHKU102. A, The doubling time ~ 33.6 min in absence of antibiotics, and B, ~36.5 min in the presence of antibiotics.
Measured by total protein assay In the measurement of the growth on plates, the total protein within the agar, which is an index of the E.coli population, was measured by (Bradford method) for a time range of 12 hours with an interval of one hour.
Fig.14 Growth curve of iHKU102 on soft agar plate (total protein). A, relation between total protein amount and OD600; B, growth curve of iHKU102 by using total protein measurement The total protein at different time points was measured (Fig.14), which is proportional to the optical density at wave length of 595nm. The first time point is before the cells were added to the agar, and put on ice. However the reading is 0.8944, rather than zero, which may be due to the protein in the LB agar. Therefore it is needed to subtract the blank from the following readings when estimating the cell population on the agar. Generally the curve increases over time, with one outliner of the third one, which also appears in the brightness result. The total protein level reaches the maximal at 10 h, with a value about twice as much as the initial protein level, and then begins to drop. Another method to measure cell growth on plate is by analysis of the brightness. This method can measure the cell density at any specific location of the plate without the need to harvest sample. However there is a drawback for this method -- variance may be due to the uneven distribution of the light reflected from the plate.
Fig.15 Relation between brightness intensity and OD value. A, photograph of plates containing inceasing cell density; B, trend line and formula indicating the relation between brightness intensity reflected from plates in panel A and OD600 value. We attempted to test if these two methods were comparable or not. So the optical analysis was conducted simultaneously with the total protein analysis at each time point. For each time point, we took a picture of the plate, and then analyzed the brightness at each pixel and added up for the total light intensity of the whole plate. Finally the brightness intensity was converted into an OD600 value and a growth curve was generated (Fig.16).
Fig.16 Growth curve of iHKU102 on soft agar plate (brightness). We next sought to measure the growth rate of iHKU102 on ring-like pattern plate. Since the total protein method is difficult to conduct in this case, we measure the brightness intensity of different region on ring-like pattern plate. In figure 17B, the brightness intensity of inner area increased in the first 15 h and then stayed at the peak in following hours. The outer area appeared at 9 h and increased gradually over time (Fig. 17C).
Fig.17 Growth curve of iHKU102 on ring-like pattern plate. By using automatic brightness measurement, Migration rate of iHKU102 was measured in the first 15 h. The migration rate can be well deducted by measuring the diameters of the inner and outer area edge. The result was 1.33 mm/h (Fig.18A). We next measured the migration rate of iHKU102 cells in agar with lower concentration (Effects of agar hardness), and found that the migration rate in 0.45 % agar (Difco) plate is 1.33 mm/h. It is much lower than that of 0.35 % agar (Difco) plate (1.53 mm/h), in which no clear ring was observed (Fig.18B; Fig.11). This data supports our hypothesis that migration rate significantly affects the development of ring-like pattern. 
Fig.18 Migration rate of iHKU102 on ring-like pattern plate Levels of LacZ and corresponding growth curves. The expression of cheZ was measured by LacZ assay. By replacing pRg in iHKU105 with pRglacZ, we generated the strain iHKU114. In this strain, the regulatory genes luxI (autoinducer synthase gene) and luxR (autoinducer receptor protein gene) are controlled by plac/area-1 and supposed to constitutively express in the presence of arabinose or IPTG; the expression of lacZ gene are driven by pluxI. Consequently, iHKU114 cells were expected to induce β-galactosidase synthesis in a cell density-dependent fashion. This was found to be the case (Fig.19A). lacZ expression was studies along with the growth curve of strains (Fig.19B). The measured LacZ activity was corrected by normalized to protein concentration and expressed in fluorescence per mg. When iHKU114 cells were inoculated into fresh medium, β-galactosidase activity decreased at the early 40 min, rose to a peak. It returned to the initial level at 70 min, and remained constant for 1.5~2 h during growth and was then induced to a level approximately 3-fold higher than preinduction levels in following 5 h. These results are consistent with the data reported previously [3], indicating a dependence on autoinducer for pluxI transcription.
Fig. 19 Levels of LacZ (panel A) and corresponding growth curves (panel B) of iHKU114 and iHKU101. To study cheZ expression in iHKU102, we replaced cheZ gene with lacZ gene and obtained the strain iHKU108. In this strain, lacZ expression is under the control of λpR-O12, which is repressed by a strong λ repressor, CI. And CI is driven by pluxI. Thus it was anticipated that λpR-O12 would be repressed in a cell density-dependent manner, and thereby lead to a decrease in the levels of β-Galactosidase activity.  Fig. 20 Level of LacZ (panel A) and corresponding growth curve (panel B) of iHKU108.
moviesTo investigate the development of the obtained patterns, a movie documenting the duration of the pattern development would bring us invaluable information to look into the mechanisms. We therefore constructed a set of photo capturing devices to record snapshots of the plates during the course of their development (movie taker). mg1655
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