Part:BBa_K1316016
constitutively expressed eGFP under the control of J23110 Anderson promoter
Constitutively expressed Enhanced Green Fluorescen Protein (eGFP). eGFP is more intense than normal GFP. The purpose of this construct is to be able to easily detect cells attached to a surface, therefore detecting biofilm formation cells. For our project that would be used for detecting curli-forming cells.
Characterization
The different constructs used are:
Different type of experiments were conducted to characterize this BioBrick:
Plate Reader
A plate reader is a machine designed to handle samples on 6-1536 well format microtiter plates for the measuring of physical properties such as absorbance, fluorescence intensity, luminescence, time-resolved fluorescence, and fluorescence polarisation.
In this module, the cells carrying the curli-forming BioBricks (CC50, CC51 or CC52) also carried a plasmid constitutively expressing eGFP (CC54). Hence, an assay to detect biofilm formation (due to the curli) can be performed. The cells can be grown on a 96-well plate, where curli formation will be induced with L-Rhamnose. The cells carrying CC50, CC51 or CC52 together with CC54 will generate curli under these conditions, whereas the cells carrying CC54 alone will not. Under the Plate reader, the wells can be analysed for green fluorescence. Before washing out the cells all wells carrying cells with CC54 should present green fluorescence. After washing out the cells, however, only the wells carrying cells with CC54 together with one of the curli-forming BioBricks should still generate green fluorescence, because of cell attachment to the walls. The final protocol developed for Plate reader analysis for this module can be found by clicking on this link .
Results - Plate Reader
Figure 1 shows the OD of the cells after two rounds of washing them out of the 96-well plate. On the image it can be appreciated that the cells carrying the curli-forming BioBricks (CC50 + CC54, CC51 + CC54 and CC52 + CC54) retain many more cells when they are induced with L-Rhamnose, whereas no noticeable increase of the OD is oserved under induction for the cells that do not carry curli-forming constructs (CC54 alone and empty cells). This suggests that cell retention happens when the curli genes are expressed.
Confocal Microscopy
Confocal microscopy is an imaging technique that allows for the visualisation of fluorescent bodies with higher resolution and improved contrast compared to Bright-field microscopy. Whereas fluorescent Bright-field microscopes excite all the sample analysed, confocal microscopes can highly reduce the excited field, thus eliminating the background noise produced by species neighbouring the body of interest.
We used confocal microscpoy technology to observe the deposition of cells at the bottom of the microscope slide. Figures 2-6 intend to represent how, after induction with L-Rhamnose, the cells forming curli are attached faster to the surface (bottom) of the microscope slide than when they are not induced.
The fact that more cells are observed at the bottom of the microscope slide for the strains carrying the CC54 plasmid alone, or the empty cells could be attributed to the fact that these cells grow faster because they do not have the burden of carrying an extra plasmid, or even two in the case of the empy cells. This idea is supported by the fact that the strains carrying curli-forming constructs (CC50, CC51 or CC52) seem to be deposited faster onto the surface of the microscope slide when they are induced than when they are not.
In order to do an objective evaluation of the number of cells, the total intensity of fluorescence of the imaged microscope slides can be done. If the fluorescence per cell of all the different strains is the same, then the more fluorescence on the microscope slide, the more cells would be attached on the surface. Then, the fluorescence per OD for each working strain was determined using the data of OD and fluorescence already available from the plate reader (the data after the 2nd cell wash was used). Figure 7 shows that all cells present the same fluorescence per OD, which makes sense as eGFP is constitutively expressed in all of them under the regulation of the same promoter. The only strain that does not make sense is the empty strain, because it is supposed to have no BioBrick and, therefore should not emit fluorescence. A possible reason for this result is the fact that LB medium was used for this assay, and it is auto-fluorescent. Another reason that could explain this is the fact that transparent 96-well plates were used for the assay. Although this well plates are supposed to be suitable for fluorescence measurements, the fact that the cells that produced fluorescence after 2 rounds of washing were, in principle, attached to the walls of the well plates could have produced noise in the measurement of neighbouring wells (producing, for instance, fluorescence noise on the wells of the empy cells). The fact that in the confocal microscope no fluorescent cells were observed supports the idea that something might have gone wrong in the measurement of fluorescence of these empty cells.
Once fluorescence per OD was measured, the total fluorescent intensity was measured per microscope slide using the ImageJ software (figure 8). According to this data, CC50 is the construct that shows more cell attachment to the slide bottom when csgA protein is induced in relation to the same non-induced strain. CC52 shows a bigger attachment improvement when induced than the CC51 construct.
Mother Machine - Widefield Fluorescence Microscopy
A Widefield Fluorescence Microscope was used to characterise the the eGFP reporter BBa_k1316016 in the Mother Machine (MM), which was used as a positive control Curli module. For more information about the Mother Machine, please visit our Microfluidics section.
MM Devices were flushed with Bovine Serum Albumin (BSA) to render the PDMS out of which the MM is made inert after plasma activation. Then cells grown in M4 minimal medium supplemented with 40mM glucose were flowed through. The M4 medium is used as a growth medium because it does not exert autofluoresence and the diameter of the cells are smaller as compared to those grown in rich media; a small diameter is required for the cells to fit in the side-channels of the MM.
The devices were then centrifuged at 3000rpm for 10 minutes, with side channels of the MM in the direction of the centripetal force. In order to coax the cells into the small channels on one side.
Unfortunately, individual cells were not found in the side channels. Reasons for this are unclear, possible causes are faulty or damaged moulds, or human error in the fabrication process. However, cells could still be imaged in the main channel, and characterised for flouresence (figure 9).
For more information about the characterisation of this construct visit the Conductive curli module characterisation on our wiki page!
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |