Part:BBa_K540000

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

This parts 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

Microscopy tests

---

In our plasmid collection described in our wiki, this part is named piG2 in the backbone Amp and piG25 in the backbone Cm. The negative control, which correspond to it, is the plasmid PUC18 and it is named piG6 in the backbone Amp. 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 strain MG1655, tagged GFP chromosomically.

For this last strain, we’d like to thank Sir 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 his work in the following publication:

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

Hence we prefered a tag GFP chromosomical and worked with the strain MG1655 in this microscopy part.

We constructed the two following strains:
- MG1655/piG2 (AmpR)
- MG1655/piG6 (AmpR)

We started a first test in M63G medium. In sterile empty plates, we introduced 10mL of M63G, we added 100μL of Amp 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 best pictures are presented here.

<img src="" width="600px">

We can easily see that the strain containing the plasmid with the part (piG2) is adherent contrary to the strain containing the plasmid without the part (piG6). We had then to determine the effect of a range of concentrations in Cobalt on the adherent strain.
</br> For this, we realized another test, using the same strains, the same medium and the same protocol.
</br> Time of incubation was shorter (15 hours) in order to see perfectly 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 strain adherent.
Again, several sites in the slides were photographed and we present here the best pictures.

</br>

<img src="" width="600px">

</br>

With these observations, we concluded that the adherence increases with the concentration in Cobalt from 0μM to 25μM. For concentrations above 25μM, the effect is cancelled.

</br>

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.

MC4100 bacteria have been transformed with this part. The transformed strain is called S26, while the negative control is called S25.

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.

The graph shown above demonstrates 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.

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 creates in new effect in transformed bacteria : cobalt makes them produce curli, in turn increasing their overall adherence.

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 47
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 2108
  
• 1000
  COMPATIBLE WITH RFC[1000]