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Results We can not wait to see this!


Measurement of LuxBrick’s Characteristic

In order to figure out the basic characteristic of k325909 submitted by Cambridge 2010, we measured growth curves of dH5a transformed k325909, which was induced by 0.3% arabinose (added from the very beginning of the incubation, and keep adding during the whole incubating process) under low temperature (25℃). As it shows in the chart, cells with and without induction grew to the logarithmic growth phase (about 5 hrs after inoculation) and stable phase (about 20 hrs after inoculation) were practically at the same time. But cells induced by arabinose displayed a significant decrease compared with those without induction.

Figure 1: growth curve of K325909 in a E.coli strain dH5a.

Detection of the Modified Protein

Detection of the Modified Protein’s Expression

Expression of mRNA for our modified protein was detected through reverse transcription PCR. The gel demonstrates that no problem exists in the transcription of the coding sequence of the modified protein lov-HTH.

The result of SDS-PAGE gel displays a satisfying expression of the modified protein lov-HTH.

Figure2: rupturing Top10 (invitrogen) cells transformed modified protein coding sequence promoted by araBAD. Extract supernatant ran the SDS-PAGE gel with PageRuler™ Prestained Protein Ladder. The electrophoretic band lies on the 24kDa line confirms the expression of the modified protein.

Detection of the Modified Protein’s Function

The result of western-blot that we used to measure the expression of GFP in cells incubated under different conditions shows that the GFP expression of cells incubated under 450nm light (37℃) is about 30% lower than cells incubated in darkness.

Figure3: rupturing BL21(DE3) cells transformed modified protein coding sequence promoted by araBAD followed by GFP promoted by ptetO (pSB1A2) incubating with and without induction of arabinose. Extract supernatant to do the western-blot. The two bands corresponding to the two samples incubated under different condition show 30% distinction.

To reassure the result of the western-bolt, we measured the relative fluorescence intensity under different gradients of IPTG induction between 0- 0.5%. The 3D diagram presents a result that the GFP expressions of cells incubated under 450nm light and in darkness are of significant difference. Moreover, the 2D diagram indicates that 0.5% IPTG induction caused the largest distinction (28%) between the light and dark among the gradients.

Figure4: Top10 (invitrogen) transformed modified protein coding sequence promoted by T7 promoter followed by GFP promoted by ptetO (pSB1A2) incubating with induction of IPTG in gradient between 0~0.5% under 450nm light (a) and in dark (b). Relative fluorescence intensity of both samples shows a decrease go with the increase of induction.

Figure5: Top10 (invitrogen) transformed modified protein coding sequence promoted by T7 promoter followed by GFP promoted by ptetO (pSB1A2) incubating with induction of 0.5% IPTG under 450nm light and in dark. The curve of relative fluorescence intensity corresponding with the cells incubated under 450nm light shows 30% lower than in dark.

The Formation of Synchronized Oscillation

The analysis of a series of images that present variations of the culture disks incubating cells transformed luxbrick following the modified protein displays a well-performed synchrony. As it can be seen in analysis diagram, above 90% of the spectrum distribution of the 2000 sampling points randomly chosen reveals a significant concentration in the cycle. The analysis result of culture disks incubating luxbrick promoted by ptetO as control indicates that with the same number of sampling points, the spectrum distribution is rarely concentrated. The result is a perfect match for the simulation.

Figure6: Spectrum analyse by randomly choosing 2000 sampling points. (a).above 90% of the sampling points’ cycles concentrate between 800 s to 1000 s. (b). the cycles of all sampling points distribute between 600 s to 2600 s. Comparing with the simulation (c), Top10(Invitrogen) transformed LuxBrick followed by the modified protein lov-HTH (pSB1A2) displays a well-performed synchrony.


Nanotube induction

1. Nanotube Induction

Figure1: Nanotube induction, with Hela cells growing under normal condition as a control group. (A) Hela cells grown under normal condition. (B) Hela cells grown under normal condition, then processed by an one-hour Cholera Toxin B induction in room temperature. (C) Hela cells grown under harsh environment simulation induction. Scale bars: all are 30 μm.

As shown in the figure above, hela cells under induction would form a significantly higher amount of nanotubes in comparison with normal hela cells. Cells underwent harsh-environmental simulation induction displayed the most prompt and radical cellular structure changes.

2. Verification for nanotube's structure

Figure2-1: Verification for nanotube's structure by immuno-staining. (A)Nucleus stained by DAPI. (B)Phalloidin staining indicating F-actin based thin nanotubes. Scale bars: all are 30 μm.

Figure2-2: Verification for nanotube's structure by immuno-staining. (A)Nucleus stained by DAPI. (B)Phalloidin staining indicating F-actin based wide nanotubes, with thin nanotubes protruded at the one end, connecting neighboring cells. Scale bars: all are 30 μm.

Two types of nanotubes have been observed: one was a wide cell protrusion-like structure that reached out from one cell and directly touched another cell even across a rather long distance; another type of nanotube was comparatively much thiner, but in a rather great number. The latter type of nanotubes could be found at the end of the former ones, thereby further connecting distant cells; or right from the middle of the cell bodies linking several neighboring cells all at once. Both types were supported by F-actin and were non-adherent to the substratum.

3. Verification for membrane continuity and communication via nanotubes.

Figure3: Verification for nanotube's structure by immuno-staining. (A)Mitochondria stained with MitoTracker indicating mitochondrial transportation between neighboring cells via nanotubes( pointed out by the arrow). (B)Two-photon Ca2+ imaging after Ca2+ dye loading indicated the interchangeable Ca2+ in nanotubes and the continuity of plasma between the connected cells . Scale bars: all are 30 μm.

Cultured cells stained with mitotracker after nanotubule induction and stabilization were then placed under microscope for observation. As it can been seen in this figure, mitochondria could travel from one cell to another via nanotube, demonstrating the property of material transportation of these induced nanotubes. Moreover, Ca2+ dye loading and two-photon Ca2+ imaging further confirmed the communication via Ca2+ flow between two connected cells.

4. Recovery after harsh-environment-simulation induction(HES induction) with further culturing and observation.

Figure4: Cellular structural changes after prolonged harsh-environment-simulation induction and recovery. (A)Cellular structural changes after five days of HES induction. Multiple nanotubes were formed during induction, resulting in a more intimate connection between distant cells. (B)Hela cells recovered in normal culture medium after HES induction. A Hela cell with unique outlook were recorded. These cells were not rare among the recovery dish. Each cluster of Hela cells had an average of one to two of these cells, stretching out their long and wide nanotubes, connecting neighboring cells. Scale bars: A:50μm; B: 10μm.

When the harsh-environmental simulation induction prolonged, cells with nanotubes underwent more radical changes in their cellular structures. After 5 days’ induction, most living cells tended to distribute most of their plasma into wide and elongated nanotubes, resulting in an octopus-like shape of each single cell and a web-like system among the whole cell colony. After incubating these cells with normal culture media for one to two days, several originally distant cells connected merely by nanotubes moved towards each other and finally clustered together, forming one big syncytium that had interchangeable plasma and organelles via nanotubes. As cells of this syncytium divided, its state remained as the newborns, with nanotube-linked neighboring cells as well. Further Ca2+ dye loading and two-photon Ca2+ imaging demonstrated a synchronization of Ca2+ flow among each individual within this syncytium( see the video below).

Electroporation and Microscopic imaging

Figure5: Microscopic imaging after electroporation. (A). Bright field image provided by confocal microscope, exhibiting some abnormal protrusion on the cell surface. (B). Same visual fields were placed under specific exciting light for Gfp emission observation. Scale bars: all are 30μm.

Cultured Hela cells after electroporation were then placed under differential interference contrast(DIC) microscope for bacterial entrance verification. Wild type E.coli K12 MG1655 expressing GFP could be seen inside the hela cells clearly. Most entered bacteria tended to cluster around the nucleus, a phenomena we reasoned that could optimize the bacterial distribution into daughter hela cells. Also, we observed a significantly higher GFP intensity given by the bacteria within hela cells, compared to bacteria stayed on the outside(data not shown), which indicate that the plasma may be a more favorable place for this facultative anaerobe to live in.

Figure5: Images indicating the transportation of wild type E.coli K12 MG1655 inside of the nanotubes. (A) 60X DIC image; (B) Gfp emission observation. Scale bars: all are 30μm.

After executed harsh-environment-simulation(HES) induction among bacteria-breeding Hela cells, symcytia similar to the former ones were obtained, with a typical spider web outlook. Visible E.coli K12 MG1655 could be seen traveling from one hela cell to another via nanotubes, indicating a non-seletivity among the transporting cargo of these nanotubule highways. However, due to the viscosity of plasma and the retardance of the cytoskeleton, E.coli K12 MG1655 within hela cells did not exhibit the mobility that we desired. In addition, the relationship between E.coli K12 MG1655 and hela cells appeared to be rather intense as usually only one of these two parties can survive after a few days of incubation of this temperal man-made endosymbiotic system.

''In our project of Bacto-Trafficking, we successfully achieved nanotube induction between Hela cells and bacterial introduction into eukaryotic cells using modified electroporation. Due to the limited time, we were not able to realize our original goal, that was to obtain distribution pattern of the bacteria within the Hela cells. But we've already observed bacteria travelling from one cell to another via nanotubes, which was very close to our expectation. Thus we believe, with a little more time, our ultimate goal of Bacto-Trafficking could be achieved.''

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