Difference between revisions of "Part:BBa K540000"

Latest revision as of 18:27, 27 October 2011

rcn-csgBAEFG, over-induces adherence in response to cobalt

This part associates the rcn promoter, whose expression increases with cobalt in the medium, with csgBAEFG, an operon that enables the production and secretion of curlis, which are adherent proteins. It contains the necessary RBS to work. With this part, strains can become hyperadherent in response to cobalt in the medium. This part may also make bacteria adherent in response to nickel, which has however not been studied.

Characterization

Following results show that this part increase the adherence ability of the transformed strain. The presence of Cobalt enhances the adherence and this response seems to be proportional to the concentration of cobalt (Cobalt range : 10µM to 100µM)

In our plasmid collection described in the wiki notebook, this part is named piG2 in the backbone Ampicillin and piG25 in the backbone Cm. The corresponding negative control is the plasmid pUC18 (=piG6 in our collection). We worked with the plasmid piG2 for the tests and we tried three different genetic backgrounds : the strain NM522 to optimize the experimental conditions, the strain MC4100 to test the effect of a range of concentrations in Cobalt and the chromosomically GFP tagged strain PHL1414 ( based on a MG1655 strain ).

For this last strain, we’d like to thank Mrs Chun Chau Sze from the NTU Laboratory who gave us the fluorescent strain and allowed us to use it for our experiment in the iGEM competition. You can find her work in the following publication: J Microbiol Methods. 2009 Feb;76(2):109-19. Epub 2008 Sep 24.

Microscopy tests

We realized a first test in a MC4100 fluorescent strain whose fluorescence had been acquired by the introduction of the plasmid p150 containing a GFP under control of a lac promoter. However, we obtained incoherent results and we had some doubts about the stability of this plasmid.

As a chromosomical GFP tag would be more reliable, these microscopy assay have been done with the PHL1414 strain, which had this characteristic.

We constructed the two following strains:
- PHL1414/piG2 (AmpR)-> STRAIN with the part Prcn-csgBAEFG in pUC19
- PHL1414/piG6 (AmpR)-> NEGATIVE CONTROL = strain with pUC18

We started a first test in M63G medium. In sterile empty plates, we introduced 10mL of M63G, we added 100μL of ampicillin (to maintain the selection pressure) and 100μL of bacteria from a saturated liquid culture. Finally, we added 3 or 4 sterile glass slides and incubated at 30°C for 23 hours. We then observed the biofilms formed at the surface of the slides with a fluorescent microscope. Several sites in the slides were photographed, the representative pictures are presented here.

Confocal microscopy

Biofilms, formed by the GFP-labeled MG1655 strains, were observed under a confocal microscope after 20 h of growth, as described below. S23 (GFP-labeled MG1655 carrying the Prcn-csgBAEFG device) and S24 (GFP-labeled MG1655 carrying the pUC18) strains were grown on glass coverslips overlayed with 15mL of MG63 medium supplemented with glucose (0,2%) and ampicilline (100 microg.L-1) in presence of cobalt (25µM). After 20h of culture at 30°C without shaking, biofilms were observed under a Zeiss confocal microscope. GFP was excited at 488 nm, and the bacterial fluorescence was collected in the range 500 to 600 nm. A 40x oil immersion objective was used for acquiring images in scanning mode. The overall three-dimensional structures of the biofilms were scanned from the solid surface to the interface with the growth medium, using a step of 1 μm. The left panel shows a 3D reconstruction done with Imaris of the poor biofilm formed by the S24 control strain. The right panel shows a 3D recontruction of the thick biofilm by the S23 strain harboring our device.

We can see that the presence of the part (strain with piG2) increase the adherence ability of the strain and leads to the formation of a thick biofilm.

We then determined the effect of a range of concentrations in Cobalt on the adherent strain.

To that end, we realized another test, using the same strains, the same medium and the same protocol.

Time of incubation was shorter (15 hours) in order to better appreciate the coverslip colonization. The range of concentrations in Cobalt was: 0 and 10μM for the negative control and 0, 10, 25, 50, 100μM for the adherent strain.
Again, several sites in the slides were photographed and we present here the representative pictures.

With these observations, we concluded that the adherence to glass increases with the Cobalt concentration from 0μM up to 25μM.

After these first qualitative results, we wanted to quantify the effect of Cobalt on the part. As we didn’t have a confocal microscope, we started adherence tests in 24 well plates.

Quantitative adherence tests in plates

In the following experiment, we have first measure the effect of the part (compared to the empty plasmid), and the effect of a range of cobalt concentration on the bacteria growth.

24-well polystyrene plates have been seeded by both strains, with increasing cobalt concentration. Cells were cultivated at 30³C in plates containing respectively 2ml of M63 minimal medium (glucose 0.2%) in each well. Both planktonic and adherent bacteria have been collected in each well and the OD600 measured. This experiment has been repeated several times, by different students.

Cobalt addition and/or presence of the synthetic operon do not impair bacterial growth.

Squares, triangles and diamonds represent the 3 replicates done for one representative experiment among the three that were performed.

These results demonstrate that total OD600 does not vary significantly with different medium conditions or strains : adding the part, or cobalt in the medium does not impact the growth of the tested bacteria. (p-value = 0.1165 in kruskal wallis test)

Biofilm formation was visualized after 48 h of culture as follows : planktonic cells were discarded and the biofilm which had developed on the bottom of the plate was washed once with M63 before drying for 1 h at 80³C. A drop of crystal violet was added for a few minutes in each well followed by extensive washes with M63, this allowed us to visualize the surface-attached bacteria. The thickness of biofilm was quantified as follows: for each well, the wash was pooled with the initial supernatant and referred to as swimming cells. The biofilm was recovered in 1 ml of M63 by scraping and pipetting up and down. The number of surface-attached and swimming bacteria was estimated from the optical density at 600 nm (OD600) to give the adherence percentage corresponding to each bacterial strain.

The synthetic operon boosts the bacterial adherence in presence of cobalt.

Squares, triangles and diamonds represent the 3 replicates done for this experiment done with the MC4100 strain as background.

From the graph above, we can draw 2 conclusions :

• A significant increase in adherence to polystyrene is observed by Co concentration ranging from 0 to 25 µM
• For higher concentrations, the adherence seems to be stable, or only slightly increasing.

Conclusion :

This part induces a synthesis of curli when cobalt is present in the medium. The low p value (P<0.0004 in anova test) indicates that exposure to cobalt has a significant effect on biofilm formation.

Further characterization

We have made fifteen preliminary 24 wells plates in order to optimize the culture condition. To that end, we had to plan their realization :

• D0 : Start of liquid precultures
• D1 : plate treatment and inoculation
• D3 : after 48 hours of incubation, revealing of the plate with OD 600 measures (biofilms and supernatants) and Crystal Violet coloration

Antibiotic response
Two 24 wells plates have been seeded with the same strain, in LB/2 medium. Ampicillin for S18 (NM522/piG2 (= the part)) has been added only in the second plate. The results were examined after 24 hours of incubation 30°C.

Qualitative approach

• BL/2 does not allow a strong adherence after 24 hours of culture
• As it can be seen on the right, adding ampicillin slightly increases the adherence of the strain, and makes it more cobalt responsive.

This can be explained by the fact that without antibiotics, the bacteria will tend to lose the part, and thus become unresponsive to cobalt.

Medium influence
During adherence tests, bacteria can be cultivated in LB/2 medium (50% Lb, 50% sterile water) or in M63G medium (100mL M63, 1ml LB, 1 ml Glucose 20%).

In order to choose the better medium, two strains have been seeded in the 2 different media, with a concentration of 10^7 cells/mL. The first is an adherent strain PHL818 (MG1655/ompR234), the second is a non-adherent strain NM522. Plates have been incubated at 30°C during 48 hours.

Results which are shown on the right are a compilation of repetitions of this experiment.

Conclusion :
A greater biofilm formation is observed using the M63G medium.

EDTA cleaning influence
Plates were washed with 4mM EDTA, and rinsed with water before growing bacteria in LB/2 medium. This aims to reduce the influence of any cobalt or nickel pre-existing in the plates, that might change the actual cobalt concentration in the plates.

Results are on the left. As can be seen, treating with EDTA beforehand slightly increases the adherence of the strain.

The stable OD proves that differences observed are not due to a difference in growth.

Conclusion :
When using this part, it is preferable to wash the plates beforehand with EDTA (or to use metal-free plastic)

Conclusion about the preliminary tests of the part :
From these experiments, we can conclude that the best conditions to grow bacteria transformed with this part are :

• Using a selective antibiotic
• M63G medium (100 mL M63 + 1 mL LB + 1 mL Glucose 20%).
• EDTA washing beforehand
• 48h growth time

Safety

This part is not toxic by itself. However, when using this part, you will probably need to handle cobalt. Cobalt is toxic in all its forms ( ionic or metallic ) by inhalation, ingestion or contact. Wear adapted personal protection equipment ( labcoat, safety glasses, safety gloves ) and dispose of it in appropriate waste containers.

Usage and Biology

This part was designed to be used in a strain with enhanced cobalt capture capacities ( like the one described in [1]). That way, the strain captures cobalt in the medium and becomes adherent, which allows it to be easily separated from the medium. Possible applications include bioremediation of radioactive cobalt in nuclear power plants, using this adherence property to build a biofilter.

[1] Bioremediation of trace cobalt from simulated spent decontamination solutions of nuclear power reactors using E. coli expressing NiCoT genes. Raghu G, Balaji V, Venkateswaran G, Rodrigue A, Maruthi Mohan P. Appl Microbiol Biotechnol. 2008 Dec.

Sequence and Features