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A vanillic acid inducible expression system for Trypanosoma brucei.

Sunter JD - Mol. Biochem. Parasitol. (2016)

Bottom Line: Reverse genetics in Trypanosoma brucei is dependent on the tetracycline inducible system for the precise control over the expression of both genes and dsRNA.Another independent inducible system for trypanosomes would enable the control of the activities of two different genes in the same cell, providing greater experimental sophistication.Here, I describe the development of the vanillic acid based inducible expression system for T. brucei, which operates independently of, and can be used in parallel with the tetracycline inducible system.

View Article: PubMed Central - PubMed

Affiliation: Sir William Dunn School of Pathology, University of Oxford, South Parks Road, OX1 3RE, UK. Electronic address: jack.sunter@path.ox.ac.uk.

No MeSH data available.


Vanillic acid inducible system is functional and independent of the tetracycline inducible system. A) Cartoon showing how the tetON and vanON inducible systems operate in T. brucei with the plasmids pDEX877 and pJ1271. B) Map of the modified pSMOX2 plasmid, pJ1173 that encodes the vanillic acid repressor protein (VanR), tetracycline repressor protein (TetR), T7 RNA polymerase (RNAP) and puromycin N-acetyl-transferase (pac). C) The sequence of the 5 VanO sites introduced into pJ1271. D) Titration of vanillic acid concentration shows that eGFP expression was induced after addition of vanillic acid and was tunable. Cells were incubated for 24 h with a range of different vanillic acid concentrations, and eGFP expression was measured by flow cytometry with 50,000 events captured per concentration. Vanillic acid was purchased from Sigma-Aldrich and dissolved in DMSO. E) and F) Comparison of eGFP expression induction by vanillic acid and doxycycline (final concentration of 1 μg/ml was used for all experiments) from pJ1271 and pDEX877. eGFP expression was induced by incubating the cells for 24 h with vanillic acid, doxycycline or both, and was measured by flow cytometry with 50000 events captured per induction. A histogram (E) and graph of normalised eGFP fluorescence intensity (FI) (F) is shown. This experiment was repeated three times for pJ1271, error bars are ±S.D. G) Growth curves for the 927 pJ1173 pJ1271 and 927 pJ1173 pDEX877 cell lines induced with vanillic acid, doxycycline or both over a 96 h period. Every 24 h the cells were split to 1 × 106 cells/ml and fresh vanillic acid and doxycycline added.
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fig0005: Vanillic acid inducible system is functional and independent of the tetracycline inducible system. A) Cartoon showing how the tetON and vanON inducible systems operate in T. brucei with the plasmids pDEX877 and pJ1271. B) Map of the modified pSMOX2 plasmid, pJ1173 that encodes the vanillic acid repressor protein (VanR), tetracycline repressor protein (TetR), T7 RNA polymerase (RNAP) and puromycin N-acetyl-transferase (pac). C) The sequence of the 5 VanO sites introduced into pJ1271. D) Titration of vanillic acid concentration shows that eGFP expression was induced after addition of vanillic acid and was tunable. Cells were incubated for 24 h with a range of different vanillic acid concentrations, and eGFP expression was measured by flow cytometry with 50,000 events captured per concentration. Vanillic acid was purchased from Sigma-Aldrich and dissolved in DMSO. E) and F) Comparison of eGFP expression induction by vanillic acid and doxycycline (final concentration of 1 μg/ml was used for all experiments) from pJ1271 and pDEX877. eGFP expression was induced by incubating the cells for 24 h with vanillic acid, doxycycline or both, and was measured by flow cytometry with 50000 events captured per induction. A histogram (E) and graph of normalised eGFP fluorescence intensity (FI) (F) is shown. This experiment was repeated three times for pJ1271, error bars are ±S.D. G) Growth curves for the 927 pJ1173 pJ1271 and 927 pJ1173 pDEX877 cell lines induced with vanillic acid, doxycycline or both over a 96 h period. Every 24 h the cells were split to 1 × 106 cells/ml and fresh vanillic acid and doxycycline added.

Mentions: Trypanosoma brucei has a well annotated genome and well developed reverse genetic tools, making it a highly tractable experimental organism used for the study of a large range of biological processes such as antigenic variation and the eukaryotic flagellum [1], [2]. Precise control over the expression of exogenous genes, mutants and dsRNA is critical for reverse genetics. In T. brucei inducible expression is controlled by the tetracycline inducible system in the tetON configuration (Fig. 1A), where tet operator (TetO) sites are located downstream of a strong promoter and the binding of tet repressor (TetR) to the TetO sites inhibits transcription from the promoter [3], [4]. Addition of tetracycline releases this inhibition by binding to the TetR, which is now unable to bind to the TetO sites, allowing transcription to occur. The successful operation of the tetracycline inducible system requires the expression of TetR; there are now several T. brucei cell lines expressing these proteins and a few examples are referenced here [4], [5], [6].


A vanillic acid inducible expression system for Trypanosoma brucei.

Sunter JD - Mol. Biochem. Parasitol. (2016)

Vanillic acid inducible system is functional and independent of the tetracycline inducible system. A) Cartoon showing how the tetON and vanON inducible systems operate in T. brucei with the plasmids pDEX877 and pJ1271. B) Map of the modified pSMOX2 plasmid, pJ1173 that encodes the vanillic acid repressor protein (VanR), tetracycline repressor protein (TetR), T7 RNA polymerase (RNAP) and puromycin N-acetyl-transferase (pac). C) The sequence of the 5 VanO sites introduced into pJ1271. D) Titration of vanillic acid concentration shows that eGFP expression was induced after addition of vanillic acid and was tunable. Cells were incubated for 24 h with a range of different vanillic acid concentrations, and eGFP expression was measured by flow cytometry with 50,000 events captured per concentration. Vanillic acid was purchased from Sigma-Aldrich and dissolved in DMSO. E) and F) Comparison of eGFP expression induction by vanillic acid and doxycycline (final concentration of 1 μg/ml was used for all experiments) from pJ1271 and pDEX877. eGFP expression was induced by incubating the cells for 24 h with vanillic acid, doxycycline or both, and was measured by flow cytometry with 50000 events captured per induction. A histogram (E) and graph of normalised eGFP fluorescence intensity (FI) (F) is shown. This experiment was repeated three times for pJ1271, error bars are ±S.D. G) Growth curves for the 927 pJ1173 pJ1271 and 927 pJ1173 pDEX877 cell lines induced with vanillic acid, doxycycline or both over a 96 h period. Every 24 h the cells were split to 1 × 106 cells/ml and fresh vanillic acid and doxycycline added.
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Related In: Results  -  Collection

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fig0005: Vanillic acid inducible system is functional and independent of the tetracycline inducible system. A) Cartoon showing how the tetON and vanON inducible systems operate in T. brucei with the plasmids pDEX877 and pJ1271. B) Map of the modified pSMOX2 plasmid, pJ1173 that encodes the vanillic acid repressor protein (VanR), tetracycline repressor protein (TetR), T7 RNA polymerase (RNAP) and puromycin N-acetyl-transferase (pac). C) The sequence of the 5 VanO sites introduced into pJ1271. D) Titration of vanillic acid concentration shows that eGFP expression was induced after addition of vanillic acid and was tunable. Cells were incubated for 24 h with a range of different vanillic acid concentrations, and eGFP expression was measured by flow cytometry with 50,000 events captured per concentration. Vanillic acid was purchased from Sigma-Aldrich and dissolved in DMSO. E) and F) Comparison of eGFP expression induction by vanillic acid and doxycycline (final concentration of 1 μg/ml was used for all experiments) from pJ1271 and pDEX877. eGFP expression was induced by incubating the cells for 24 h with vanillic acid, doxycycline or both, and was measured by flow cytometry with 50000 events captured per induction. A histogram (E) and graph of normalised eGFP fluorescence intensity (FI) (F) is shown. This experiment was repeated three times for pJ1271, error bars are ±S.D. G) Growth curves for the 927 pJ1173 pJ1271 and 927 pJ1173 pDEX877 cell lines induced with vanillic acid, doxycycline or both over a 96 h period. Every 24 h the cells were split to 1 × 106 cells/ml and fresh vanillic acid and doxycycline added.
Mentions: Trypanosoma brucei has a well annotated genome and well developed reverse genetic tools, making it a highly tractable experimental organism used for the study of a large range of biological processes such as antigenic variation and the eukaryotic flagellum [1], [2]. Precise control over the expression of exogenous genes, mutants and dsRNA is critical for reverse genetics. In T. brucei inducible expression is controlled by the tetracycline inducible system in the tetON configuration (Fig. 1A), where tet operator (TetO) sites are located downstream of a strong promoter and the binding of tet repressor (TetR) to the TetO sites inhibits transcription from the promoter [3], [4]. Addition of tetracycline releases this inhibition by binding to the TetR, which is now unable to bind to the TetO sites, allowing transcription to occur. The successful operation of the tetracycline inducible system requires the expression of TetR; there are now several T. brucei cell lines expressing these proteins and a few examples are referenced here [4], [5], [6].

Bottom Line: Reverse genetics in Trypanosoma brucei is dependent on the tetracycline inducible system for the precise control over the expression of both genes and dsRNA.Another independent inducible system for trypanosomes would enable the control of the activities of two different genes in the same cell, providing greater experimental sophistication.Here, I describe the development of the vanillic acid based inducible expression system for T. brucei, which operates independently of, and can be used in parallel with the tetracycline inducible system.

View Article: PubMed Central - PubMed

Affiliation: Sir William Dunn School of Pathology, University of Oxford, South Parks Road, OX1 3RE, UK. Electronic address: jack.sunter@path.ox.ac.uk.

No MeSH data available.