Team:Guanajuato Mx/CompositeParts

iGEM Guanajuato Mx

COMPOSITE PARTS


LasR under the regulation of the promoter chiA74 (BBa_K2057003)

The promoter chiA74 from Bacillus thuringiensis is functional in Escherichia coli, and regulate the expression of a gene in a constitutive form. This composite part is formed from the biobricks BBa_K2057000 (promoter chiA74), BBa_C0079 (lasR gene).

References

  1. Barboza-Corona JE, Delgadillo-Ángeles JL, Castañeda-Ramírez JC, Barboza- Pérez UE, Casados-Vázquez LE, Bideshi DK, del Rincón-Castro MC (2014) Bacillus thuringiensis subsp. kurstaki HD1 as a factory to synthesize alkali-labile ChiA74∆sp chitinase inclusions, Cry crystals and spores for applied use. Microbial Cell Factories. 13:15. doi:10.1186/1475-2859-13-15
  2. Barboza-Corona JE, Nieto-Mazzocco E, Velázquez-Robledo R, Salcedo- Hernández R, Bautista M, Jiménez B, Ibarra JE (2003) Cloning, sequencing and expression of the chitinase gene chiA74 from Bacillus thuringiensis. Applied and Environmental Microbiology. 69(2): 1023-1029.
  3. Saeidi N, Wong CK, Lo TM, Nguyen HX, Ling H, Leong SS, Poh CL, Chang MW (2011) Engineering microbes to sense and eradicate Pseudomonas aeruginosa, a human pathogen. Mol Syst Biol. 7:521


LasR under the regulation of the promoter BtI-BtII (BBa_K2057004)

The promoter BtII from Bacillus thuringiensis regulate the expression of the cry genes during sporulation, but it is functional in Escherichia coli in a constitutive form. As promoter is not available in the biobricks, we amplified, sequenced and characterized using the cry1Ac gene as template. Promoter BtI-BtII also includes the ribosome binding site. This composite part is formed from the biobricks BBa_K2057001 (promoter BtI-BtII), BBa_C0079 (lasR gene).

References

  1. Von Tersch MA, Robbins HL, Jany CS, Johnson TB (1991) Insecticidal toxins from Bacillus thuringiensis subsp. kenyae: gene cloning and characterization and comparison with B. thuringiensis subsp. kurstaki CryIA(c) toxins Appl. Environ. Microbiol. 57 (2): 349-358.
  2. Sedlak M, Walter T, Aronson A (2000) Regulation by overlapping promoters of the rate of synthesis and deposition into crystalline inclusions of Bacillus thuringiensis delta-endotoxins. J Bacteriol. 182(3):734-41.


Expression of gfp under the regulation of promoter ChiA74 (BBa_K2057005)

We made this composite part to check the functionality of the promoter chiA74 in Escherichia coli. Biobrick BBa_K2057000 (promoter chiA74) includes the promoter sequence and the ribosome binding sequence. The gfp gene is obtained from BBa_E0040.

References

  1. Barboza-Corona JE, Delgadillo-Ángeles JL, Castañeda-Ramíırez JC, Barboza- Pérez UE, Casados-Vázquez LE, Bideshi DK and del Rincón-Castro MC (2014) Bacillus thuringiensis subsp. kurstaki HD1 as a factory to synthesize alkali-labile ChiA74∆sp chitinase inclusions, Cry crystalsand spores for applied use. Microb Cell Fact 13, 15. doi: 10.1186/1475-2859-13-15
  2. Barboza Corona JE, Nieto-Mazzocco E, Velázquez-Robledo R, Salcedo- Hernandez R, Bautista M, Jiménez B, Ibarra JE (2003) Cloning, sequencing and expression of the chitinase chiA74 from Bacillus thuringiensis. Appl Environ Microbiol. 2003 Feb;69(2):1023-1029.


Expression of gfp under the regulation of promoter BtI-BtII (BBa_K2057006)

We made this composite part to check the functionality of the promoter BtI-BtII in Escherichia coli. Biobrick BBa_K2057001 (promoter BtI-BtII) includes the promoter sequence and the ribosome binding sequence. The gfp gene is obtained from BBa_E0040.

As promoter BtI-BtII (BtI-BtIIp) was obtained from a gene found in B. thuringiensis, we want to test whether it drives the expression of a gene in E.coli. Our purpose was to demonstrate if promoters BtI-BtII and chiA74 could be used as constitutive promoters to express the LasR in our biological circuit. E. coli Top10/pHT3101- chiA74p, E. coli Top10/pHT3101-BtI-BtIIp, and E. coli Top10/pHT3101-BtI-BtIIp-gfp were grown at 37ºC to reach approximately 1 X 108 cells/mL. Cells were centrifuged, dissolved in 150 mM NaCl, 100 mM Tris, pH8, and sonicated. Protein concentration was determined by the Bradford method (BioRad). Fluorescence was determined using the same amount of protein. As was expected, the higher fluorescence, due to the GFP, was observed with E. coli Top10/pHT3101-BtI-BtIIp- gfp, indicating that dual promoter BtI-BtII from B. thuringiensis is functional in E. coli.

References

  1. Von Tersch MA, Robbins HL, Jany CS, Johnson TB (1991) Insecticidal toxins from Bacillus thuringiensis subsp. kenyae: gene cloning and characterization and comparison with B. thuringiensis subsp. kurstaki CryIA(c) toxins Appl. Environ. Microbiol. 57 (2): 349-358.
  2. Sedlak M1, Walter T, Aronson A (2000) Regulation by overlapping promoters of the rate of synthesis and deposition into crystalline inclusions of Bacillus thuringiensis delta-endotoxins. J Bacteriol. 182(3):734-41.


Alginate lyase and microcin controlled by lasI promoter (BBa_K2057008)

The alignate lyase gen and the microcin are controlled the lasI gene, which sense the LasR-homoserinelactone.

Alginate lyase come from the chromosome of Bacillus thuringiensis 4Q7. For making this part the next biobricks are used: BBa_K649000 (lasI promoter), BBa_K2057007 (chiA74 signal peptide), BBa_K2057002 (alginate lyase gene) and BBa_K565004 (microcin).

References

  1. Farrel EK, Tipton PA (2012) Functional characterization of AlgL, an alginate lyase from Pseudomona aeruginosa. Biochem. 51:10259-10266.
  2. Saeidi N, Wong CK, Lo TM, Nguyen HX, Ling H, Leong SS, Poh CL, Chang MW (2011) Engineering microbes to sense and eradicate Pseudomonas aeruginosa, a human pathogen. Mol Syst Biol. 7:521
  3. Barboza Corona JE, Nieto-Mazzocco E, Velázquez-Robledo R, Salcedo- Hernandez R, Bautista M, Jiménez B, Ibarra JE (2003) Cloning, sequencing and expression of the chitinase chiA74 from Bacillus thuringiensis. Appl Environ Microbiol. 2003 Feb;69(2):1023-1029.
  4. Zschüttig A, Zimmermann K, Blom J, Goesmann A, Pöhlmann C (2012) Identification and characterization of Microcin S, a new antibacterial peptide produced by probiotic Escherichia coli G3/10. PLoS ONE 7(3): e33351. doi:10.1371/journal.pone.0033351


Alginate lyase controlled by lasI promoter (BBa_K2057009)

The alignate lyase gene is controlled the lasI promoter, which sense the LasR- homoserinelactone. Secretion of alginate lyase is controlled by the signal peptide of chiA74.

References

  1. Farrel EK, Tipton PA (2012) Functional characterization of AlgL, an alginate lyase from Pseudomona aeruginosa. Biochem. 51:10259-10266.
  2. Saeidi N, Wong CK, Lo TM, Nguyen HX, Ling H, Leong SS, Poh CL, Chang MW (2011) Engineering microbes to sense and eradicate Pseudomonas aeruginosa, a human pathogen. Mol Syst Biol. 7:521
  3. Barboza Corona JE, Nieto-Mazzocco E, Velázquez-Robledo R, Salcedo- Hernandez R, Bautista M, Jiménez B, Ibarra JE (2003) Cloning, sequencing and expression of the chitinase chiA74 from Bacillus thuringiensis. Appl Environ Microbiol. 2003 Feb;69(2):1023-1029.


iGEM Guanajuato Mx

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