DNA

Part:BBa_K3791025

Designed by: Laura Snchez Ruiz, Auba Fuster Pal   Group: iGEM21_UPF_Barcelona   (2021-10-12)


Construct for modulating protein E transcription

This part is a construct intended to permit both the transcription and the repression of the gene that encodes for protein E. Such protein has an autolytic function: its aim is to lysate the host bacteria by a not exactly determined mechanism of action (see BBa_K2500006). This event results in the release of the cytoplasmic content and the consequent cell death.

The full construct is formed by a promoter, a ribosome binding site, the protein E gene and finally a terminator. The first component is pBAD-araC promoter (BBa_I0500), which is composed of several functional elements, being the pBAD promoter and pC promoter. These have a functional relationship susceptible to the nutrients found in the medium culture [1].

If there’s absence of glucose but plenty of L-arabinose, the latter will bind to the AraC protein promoting its dimerization. The complex will bind to the l1 and l2 sites of the operator site found between pBAD and pC, inducing binding of CAP (cyclic AMP receptor protein) and facilitating transcription in both promoters. This creates a positive loop for AraC transcription, as well as for the gene found to be regulated by pBAD [2]. Otherwise, if the nutrient found in the medium is glucose, AraC will dimerize without arabinose, binding to the O2 and I2 regions of the operon and inducing looping of DNA [3]. Also, plasmatic cAMP will decrease preventing CAP from binding the regulating region, thus transcription will be down-regulated in a negative loop.

The other pieces of this part are, apart from the aforementioned protein E, the following efficient RBS and the E. coli his operon terminator: BBa_K2306014 and BBa_B0053.

Figure 1. Protein E construct, constituted by pBAD AraC promoter, RBS, Protein E and His operon terminator.

Figure 1 Description: The construct structure is represented in a simple diagram. The AraC pBAD functional element is placed on the 5’ end, followed by the RBS, the gene codifying for Protein E, and His operon terminator on the 3’ end. The AraC codifying region is placed upstream of pBAD, but it’s transcribed in the opposite direction.


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 81
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

This part is used inside NZY5α competent cells, where we transform our cloned plasmid containing Protein E controlled by the pBAD promoter. For the cloning, we used Gibson Assembly. This method takes advantage of adjacent DNA fragments that have complementary ends, to fuse two different parts together [4]. More details about the building process are available on our Biosensor library building page.

One of the simplest and cheapest ways to visually and quantitatively verify that cells have been lysed is by Bradford's assay. It is a colorimetric assay based on a dye called Coomassie Brilliant Blue G-250, that can bind protein complexes. In an acidic environment and after the addition of the proteins, there is a shift from the reddish-brown color of the dye to its bluish form. This change can be observed at naked eye or by measuring the absorbance at 595nm with a Spectrophotometer. Finally, to quantify the amount of protein in the compound, the measurements can be extrapolated to a BSA standard curve (blank) [5].


Figure 2. Bradford Assay.

Figure 2 Description: Representation of the Bradford Assay. Scheme of colorimetric change from brown to blue after the addition of a protein complex, quantitative measurement and extrapolation of absorbance to a BSA standard curve.


Characterization

For this section, preliminary results which give important information were accomplished. We also realized some crucial issues for our further work in the future. Regarding the autolysis protein, we were able to perform the cloning of the fragments correctly (Figure 3). As can be seen, both the Digestion and PCR of the purified parts went well, and we assume that there was no problem in performing the Gibson Assembly.

Figure 3. Electrophoresis gel with PCR and Digestion results.

Figure 3 Description: An electrophoresis gel is displayed. The band in the center corresponds to the PCR result while the one on the right corresponds to the digestion result. On the left is the DNA ladder.

However, we did not consider an important aspect. After performing a transformation on NZY5α competent cells, we cultured them on an Agar plate without D-Glucose. The result was that the next day we had not obtained any colony (Figure 4).

Figure 4. Empty plate after transformation with plasmid containing codifying prot.E fragment.

Figure 4 Description: This is an empty LB Agar plate. It is labeled "Autolysis Protein E ChloR 14.10.21 150μL (no glucose) induction L-ARABINOSE".


Conclusion
We consider that the lack of colonies is due to the leakiness of the expression of the autolysis protein and that its minimal expression is already able to lyse the cells. Therefore, we know that in the future we will have to grow the cells in a medium rich in D-glucose to completely inhibit the PBAD promoter [6]. Further experiments remain to be performed, but we have proved one important feature about Protein E leakiness.


References

[1] Gearing, M. (2018, January 18). Plasmids 101: Inducible Promoters. Blog Addgene. https://blog.addgene.org/plasmids-101-inducible-promoters
[2] Martin, K., Huo, L., & Schleif, R. F. (1986). The DNA loop model for ara repression: AraC protein occupies the proposed loop sites in vivo and repression-negative mutations lie in these same sites. Proceedings of the National Academy of Sciences, 83(11), 3654–3658. https://doi.org/10.1073/pnas.83.11.3654
[3] Lobell, R. B., & Schleif, R. F. (1990). DNA Looping and Unlooping by AraC Protein. Science, 250(4980), 528–532. https://doi.org/10.1126/science.2237403
[4] Gibson, D. G., Young, L., Chuang, R.-Y., Venter, J. C., Hutchison, C. A., 3rd, & Smith, H. O. (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods, 6(5), 343–345 doi: 10.1038/nmeth.1318
[5] Bradford Assay (Bradford Reagent) - ES. (n.d.). Retrieved October 19, 2021, from: Thermofisher
[6] Guzman, L. M., Belin, D., Carson, M. J., & Beckwith, J. (1995). Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. Journal of Bacteriology, 177(14), 4121–4130. Doi: 10.1128/jb.177.14.4121-4130.1995

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