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Construction of three new Gateway® expression plasmids for Trypanosoma cruzi.

Alonso VL, Ritagliati C, Cribb P, Serra EC - Mem. Inst. Oswaldo Cruz (2014)

Bottom Line: Both plasmids were assayed by introducing green fluorescent protein (GFP) by recombination and the integrity of the double-tagged protein was determined by western blotting and immunofluorescence microscopy.The third Gateway adapted vector assayed was the inducible pTcINDEX.When tested with GFP, pTcINDEX-GW showed a good response to tetracycline, being less leaky than its precursor (pTcINDEX).

View Article: PubMed Central - PubMed

Affiliation: Facultad de Ciencias Bioqu├şmicas y Farmac├ęuticas, Universidad Nacional de Rosario, Rosario, Argentina.

ABSTRACT
We present here three expression plasmids for Trypanosoma cruzi adapted to the Gateway® recombination cloning system. Two of these plasmids were designed to express trypanosomal proteins fused to a double tag for tandem affinity purification (TAPtag). The TAPtag and Gateway® cassette were introduced into an episomal (pTEX) and an integrative (pTREX) plasmid. Both plasmids were assayed by introducing green fluorescent protein (GFP) by recombination and the integrity of the double-tagged protein was determined by western blotting and immunofluorescence microscopy. The third Gateway adapted vector assayed was the inducible pTcINDEX. When tested with GFP, pTcINDEX-GW showed a good response to tetracycline, being less leaky than its precursor (pTcINDEX).

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: restriction maps and genetic elements of the Gateway┬« plasmids (A)pTEX-TAPtag-GW map. The ampicillin resistance gene (neor), Gateway┬« cassette and TAPtag sequence are flanked by the5ÔÇÖ-upstream and 3-downstream regions of the Trypanosoma cruziglycosomal glycosomal glyceraldehyde-3-phosphate dehydrogenase gene (gGAPDH)genes. The Gateway┬« cassette consists of the attR1 and R2 sites (black boxes), thechloramphenicol resistance gene (Cmr) and the ccdB gene. TheTAPtag cassette consists of the calmodulin-binding peptide (CBP), protein A (PA)and a spacer region with a Tobacco Etch Virus (TEV) protease cleavage site; B:pTREX-TAPtag-GW map. This vector has the same features as pTEX with the additionof a ribosomal promoter (with a transcription starting point, black arrow head)and the HX1 fragment; C: inducible expression vector pTcINDEX-GW.The grey box indicates the HX1 fragment. The T. cruzi actinintergenic region (TcActin IG), the T7 transcriptional terminator (T) and theGateway┬« cassette are shown. The black flags indicate the T7 promoter and the greycircle denotes the location of the tetracycline operator. R-NTS/P is the ribosomalnon-transcribed spacer and promoter region used for targeting. The Roman numeralsI and II indicate the two halves of the targeting sequence cloned in the oppositeorientation of that in the genome. The black arrowhead indicates the location ofthe polymerase I transcription start site.
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f01: : restriction maps and genetic elements of the Gateway┬« plasmids (A)pTEX-TAPtag-GW map. The ampicillin resistance gene (neor), Gateway┬« cassette and TAPtag sequence are flanked by the5ÔÇÖ-upstream and 3-downstream regions of the Trypanosoma cruziglycosomal glycosomal glyceraldehyde-3-phosphate dehydrogenase gene (gGAPDH)genes. The Gateway┬« cassette consists of the attR1 and R2 sites (black boxes), thechloramphenicol resistance gene (Cmr) and the ccdB gene. TheTAPtag cassette consists of the calmodulin-binding peptide (CBP), protein A (PA)and a spacer region with a Tobacco Etch Virus (TEV) protease cleavage site; B:pTREX-TAPtag-GW map. This vector has the same features as pTEX with the additionof a ribosomal promoter (with a transcription starting point, black arrow head)and the HX1 fragment; C: inducible expression vector pTcINDEX-GW.The grey box indicates the HX1 fragment. The T. cruzi actinintergenic region (TcActin IG), the T7 transcriptional terminator (T) and theGateway┬« cassette are shown. The black flags indicate the T7 promoter and the greycircle denotes the location of the tetracycline operator. R-NTS/P is the ribosomalnon-transcribed spacer and promoter region used for targeting. The Roman numeralsI and II indicate the two halves of the targeting sequence cloned in the oppositeorientation of that in the genome. The black arrowhead indicates the location ofthe polymerase I transcription start site.

Mentions: Then, the Gateway┬« cassette was PCR-amplified from the pDESTÔäó17 vector(Invitrogen) and inserted into EcoRV-HindIII sites (Fig. 1A, B). Todetermine the functionality of pTEX-TAPtag-GW and pTREX-TAPtag-GW, the green fluorescentprotein (GFP) sequence was transferred by recombination (LR) with the LRClonase┬« II enzyme mix (Invitrogen) into the TAPtag-GW plasmids as describedby the manufacturer from the previously constructed pENTR3C-GFP vector. The plasmids weremaintained in the DB3.1 Escherichia coli strain.


Construction of three new Gateway® expression plasmids for Trypanosoma cruzi.

Alonso VL, Ritagliati C, Cribb P, Serra EC - Mem. Inst. Oswaldo Cruz (2014)

: restriction maps and genetic elements of the Gateway┬« plasmids (A)pTEX-TAPtag-GW map. The ampicillin resistance gene (neor), Gateway┬« cassette and TAPtag sequence are flanked by the5ÔÇÖ-upstream and 3-downstream regions of the Trypanosoma cruziglycosomal glycosomal glyceraldehyde-3-phosphate dehydrogenase gene (gGAPDH)genes. The Gateway┬« cassette consists of the attR1 and R2 sites (black boxes), thechloramphenicol resistance gene (Cmr) and the ccdB gene. TheTAPtag cassette consists of the calmodulin-binding peptide (CBP), protein A (PA)and a spacer region with a Tobacco Etch Virus (TEV) protease cleavage site; B:pTREX-TAPtag-GW map. This vector has the same features as pTEX with the additionof a ribosomal promoter (with a transcription starting point, black arrow head)and the HX1 fragment; C: inducible expression vector pTcINDEX-GW.The grey box indicates the HX1 fragment. The T. cruzi actinintergenic region (TcActin IG), the T7 transcriptional terminator (T) and theGateway┬« cassette are shown. The black flags indicate the T7 promoter and the greycircle denotes the location of the tetracycline operator. R-NTS/P is the ribosomalnon-transcribed spacer and promoter region used for targeting. The Roman numeralsI and II indicate the two halves of the targeting sequence cloned in the oppositeorientation of that in the genome. The black arrowhead indicates the location ofthe polymerase I transcription start site.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4325611&req=5

f01: : restriction maps and genetic elements of the Gateway┬« plasmids (A)pTEX-TAPtag-GW map. The ampicillin resistance gene (neor), Gateway┬« cassette and TAPtag sequence are flanked by the5ÔÇÖ-upstream and 3-downstream regions of the Trypanosoma cruziglycosomal glycosomal glyceraldehyde-3-phosphate dehydrogenase gene (gGAPDH)genes. The Gateway┬« cassette consists of the attR1 and R2 sites (black boxes), thechloramphenicol resistance gene (Cmr) and the ccdB gene. TheTAPtag cassette consists of the calmodulin-binding peptide (CBP), protein A (PA)and a spacer region with a Tobacco Etch Virus (TEV) protease cleavage site; B:pTREX-TAPtag-GW map. This vector has the same features as pTEX with the additionof a ribosomal promoter (with a transcription starting point, black arrow head)and the HX1 fragment; C: inducible expression vector pTcINDEX-GW.The grey box indicates the HX1 fragment. The T. cruzi actinintergenic region (TcActin IG), the T7 transcriptional terminator (T) and theGateway┬« cassette are shown. The black flags indicate the T7 promoter and the greycircle denotes the location of the tetracycline operator. R-NTS/P is the ribosomalnon-transcribed spacer and promoter region used for targeting. The Roman numeralsI and II indicate the two halves of the targeting sequence cloned in the oppositeorientation of that in the genome. The black arrowhead indicates the location ofthe polymerase I transcription start site.
Mentions: Then, the Gateway┬« cassette was PCR-amplified from the pDESTÔäó17 vector(Invitrogen) and inserted into EcoRV-HindIII sites (Fig. 1A, B). Todetermine the functionality of pTEX-TAPtag-GW and pTREX-TAPtag-GW, the green fluorescentprotein (GFP) sequence was transferred by recombination (LR) with the LRClonase┬« II enzyme mix (Invitrogen) into the TAPtag-GW plasmids as describedby the manufacturer from the previously constructed pENTR3C-GFP vector. The plasmids weremaintained in the DB3.1 Escherichia coli strain.

Bottom Line: Both plasmids were assayed by introducing green fluorescent protein (GFP) by recombination and the integrity of the double-tagged protein was determined by western blotting and immunofluorescence microscopy.The third Gateway adapted vector assayed was the inducible pTcINDEX.When tested with GFP, pTcINDEX-GW showed a good response to tetracycline, being less leaky than its precursor (pTcINDEX).

View Article: PubMed Central - PubMed

Affiliation: Facultad de Ciencias Bioqu├şmicas y Farmac├ęuticas, Universidad Nacional de Rosario, Rosario, Argentina.

ABSTRACT
We present here three expression plasmids for Trypanosoma cruzi adapted to the Gateway® recombination cloning system. Two of these plasmids were designed to express trypanosomal proteins fused to a double tag for tandem affinity purification (TAPtag). The TAPtag and Gateway® cassette were introduced into an episomal (pTEX) and an integrative (pTREX) plasmid. Both plasmids were assayed by introducing green fluorescent protein (GFP) by recombination and the integrity of the double-tagged protein was determined by western blotting and immunofluorescence microscopy. The third Gateway adapted vector assayed was the inducible pTcINDEX. When tested with GFP, pTcINDEX-GW showed a good response to tetracycline, being less leaky than its precursor (pTcINDEX).

Show MeSH
Related in: MedlinePlus