Difference between revisions of "Part:BBa K540000"

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−	<p>In our plasmid collection described in the wiki, <b>this part is named piG2</b> in the backbone Ampicillin	+	<p>In our plasmid collection described in the wiki notebook, <b>this part is named piG2</b> in the backbone Ampicillin
	and piG25 in the backbone Cm. The corresponding <b>negative control</b> is the plasmid PUC18 and it <b>is named piG6</b> in the backbone Ampicillin.		and piG25 in the backbone Cm. The corresponding <b>negative control</b> is the plasmid PUC18 and it <b>is named piG6</b> in the backbone Ampicillin.
	We worked with the plasmid piG2 for the tests and we tried three different genetic		We worked with the plasmid piG2 for the tests and we tried three different genetic

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Revision as of 12:16, 21 September 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 adherent 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 enhance 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 and it is named piG6 in the backbone Ampicillin. 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 MG1655.

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

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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. 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 MG1655 strain.

We constructed the two following strains:
- MG1655/piG2 (AmpR)-> STRAIN with the part Prcn-csgBAEFG
- MG1655/piG6 (AmpR)-> NEGATIVE CONTROL

We started a first test in M63G medium. In sterile empty plates, we introduced 10mL of M63G, we added 100μL of Ampicillin 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.

We can see that the presence of the part (strain with piG2) increase the adherence ability of the strain.

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 see the effect of Cobalt on the strain. 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 increases with the Cobalt concentration from 0μM up to 25μM. For concentrations above 25μM, the effect of the cobalt is not visible anymore.

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

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Useful development to choose the best adherence test conditions are discribed in the experiences tab

MC4100 bacteria have been transformed with the Prcn-csgBAEFG part. We constructed the two following strains:
- MC4100/piG2 (AmpR)-> STRAIN with the part Prcn-csgBAEFG
- MC4100/piG6 (AmpR)-> NEGATIVE CONTROL

In the following experiment, we are testing both the effect of this part on the bacteria, and the response to cobalt with and without the added part.

24-well plates have been seeded by both strain, with increasing cobalt concentration. This experiment has been repeated several times, by different students. Total OD measurement shows the relative growth of the bacteria in the different well, while the OD measurement of the fixed fraction of bacteria will reveal the effect of the part on biofilm formation.

Squares, triangles and diamonds represent the 3 replicates done for this experiment

These results demonstrate that total OD does not vary significantly with different medium conditions.

This implies that adding the part, or cobalt in the medium does not impact the growth of the tested bacteria, and that differences in OD between wells will be significant.

Squares, triangles and diamonds represent the 3 replicates done for this experiment

From the graph above, we can draw 2 conclusions :

• A significant increase in adherence 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.

Further characterization

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Antibiotic response
Two 24 wells plates have been seeded with the same strains, in LB/2 medium. Ampicilline for S18 and Cm have been added only in the second plate. The results have examined after 24 hours of incubation 30°C.

Qualitative approach
As can be seen on the right, adding ampicilline boosts the adherence of the strain, and makes it more cobalt responsive.

Quantitative approach
Furthermore, the adherence level was determined quantitatively using OD measurements. Resulting graphs are shown on the left.

P1 indicates the first plate, without antibiotics, while P2 indicates the second plate, with antibiotics.

Conclusion: it can clearly be seen that while the growth of the bacteria are similar, as they have similar total OD, adding the antibiotics makes them a lot more adherent, and enable the cobalt-response.

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 grown 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, the second is a non-adherent strain NM522. Plates have been incubated at 30°C during 48 hours.

Results which are show 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. 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 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