Construction of a short-lived fluorescent protein transcriptional reporter
Publication Date
July 2019
Location
LSU Health Medical Education Building
Document Type
Abstract
Start Date
26-7-2019 9:00 AM
End Date
26-7-2019 12:00 PM
Description
Chlamydia trachomatis is an obligate intracellular Gram-negative bacterium that causes various acute and chronic diseases, including eye infections, trachoma, genital infections and the more invasive sexually transmitted infection; lymphogranuloma venereum (LGV). Despite the increased awareness and intensified testing of the C. trachomatis diseases, a rising number of new infections has been reported. Its high contagion and detrimental sequel have continued to be a global health burden. A better understanding of the C. trachomatis biology is critical for the development of new therapy against C. trachomatis diseases. Studies of the unique chlamydial developmental cycle and gene expression have been greatly facilitated by recent success in the development of fluorescence protein reporter gene using transformation of C. trachomatis with a plasmid. This powerful tool allows to directly visualize dynamic growth of Chlamydia in situ in individual living cells. We have shown that a red-shift green fluorescence protein (rsGFP) driven by a chlamydial ompA promoter has a half live of ~3 hours in a transformed C. trachomatis LGV L2 strain under the condition that results in a productive infection. An ideal reporter gene of temporal transcription programs includes a short half-life that avoids extended accumulation when transcription is turning off. In an effort to meet this criterion, here, we adapted a GFP variant designated as GFP[LVA] which contained a short peptide sequence to the C-terminal end of intact GFP and rendered GFP’s sensitivity to the action of endogenous tail-specific protease, tsp, in E. coli. C. trachomatis encodes a homologue of Tsp. To test the hypothesis that proteolytic degradation of GFP[LVA] is a conserved trait in C. trachomatis, we constructed a new reporter plasmid pCtGFP[LVA] for chlamydial transformation. Briefly, E. coli strain DH10β cells (New England Biolabs) were used for the molecular cloning. A two-step cloning strategy was used to construct vector pCtGFP[LVA]. First, the pPeuo[LVA]E was constructed by ligation of SpeI/BstBI digested pBC¬A1-009 (this vector contains gene encoding GFP[LVA]) and the SpeI/BstBI-elevated fragment from pPvGFP:SW2. The latter fragment carries a chlamydial promoter of euo flanked by a ribosomal binding site of tuf gene. Second, the SpeI and SmaI–digested DNA fragment of pPeuo[LVA]E was inserted into the SpeI-(Sal-blunted)-digested pBOMBeuo. Therefore, the resultant plasmid pCtGFP[LVA] contains gene coding for GFP[LVA]) under the control of an early promoter from chlamydial euo gene as well as eight open reading frames (ORFs) and an origin of replication from conserved C. trachomatis plasmid. This plasmid also contains an E. coli origin of replication and a bla gene encoding resistance to beta-lactam antibiotics allowing for selection of recombinants with ampicillin. We are presently working on transformation of C. trachomatis with pCtGFP[LVA] and determining 1) what is the half-life in C. trachomatis, and 2) whether the short-lived GFP[LVA] reporter gene could accurately track and predict the transient mRNA profile of the early chlamydial gene, euo during infection.
Recommended Citation
Hassan, Hassan A., "Construction of a short-lived fluorescent protein transcriptional reporter" (2019). Summer Research Internship Program. 14.
https://digitalscholar.lsuhsc.edu/srip/2019/undergrad/14
Construction of a short-lived fluorescent protein transcriptional reporter
LSU Health Medical Education Building
Chlamydia trachomatis is an obligate intracellular Gram-negative bacterium that causes various acute and chronic diseases, including eye infections, trachoma, genital infections and the more invasive sexually transmitted infection; lymphogranuloma venereum (LGV). Despite the increased awareness and intensified testing of the C. trachomatis diseases, a rising number of new infections has been reported. Its high contagion and detrimental sequel have continued to be a global health burden. A better understanding of the C. trachomatis biology is critical for the development of new therapy against C. trachomatis diseases. Studies of the unique chlamydial developmental cycle and gene expression have been greatly facilitated by recent success in the development of fluorescence protein reporter gene using transformation of C. trachomatis with a plasmid. This powerful tool allows to directly visualize dynamic growth of Chlamydia in situ in individual living cells. We have shown that a red-shift green fluorescence protein (rsGFP) driven by a chlamydial ompA promoter has a half live of ~3 hours in a transformed C. trachomatis LGV L2 strain under the condition that results in a productive infection. An ideal reporter gene of temporal transcription programs includes a short half-life that avoids extended accumulation when transcription is turning off. In an effort to meet this criterion, here, we adapted a GFP variant designated as GFP[LVA] which contained a short peptide sequence to the C-terminal end of intact GFP and rendered GFP’s sensitivity to the action of endogenous tail-specific protease, tsp, in E. coli. C. trachomatis encodes a homologue of Tsp. To test the hypothesis that proteolytic degradation of GFP[LVA] is a conserved trait in C. trachomatis, we constructed a new reporter plasmid pCtGFP[LVA] for chlamydial transformation. Briefly, E. coli strain DH10β cells (New England Biolabs) were used for the molecular cloning. A two-step cloning strategy was used to construct vector pCtGFP[LVA]. First, the pPeuo[LVA]E was constructed by ligation of SpeI/BstBI digested pBC¬A1-009 (this vector contains gene encoding GFP[LVA]) and the SpeI/BstBI-elevated fragment from pPvGFP:SW2. The latter fragment carries a chlamydial promoter of euo flanked by a ribosomal binding site of tuf gene. Second, the SpeI and SmaI–digested DNA fragment of pPeuo[LVA]E was inserted into the SpeI-(Sal-blunted)-digested pBOMBeuo. Therefore, the resultant plasmid pCtGFP[LVA] contains gene coding for GFP[LVA]) under the control of an early promoter from chlamydial euo gene as well as eight open reading frames (ORFs) and an origin of replication from conserved C. trachomatis plasmid. This plasmid also contains an E. coli origin of replication and a bla gene encoding resistance to beta-lactam antibiotics allowing for selection of recombinants with ampicillin. We are presently working on transformation of C. trachomatis with pCtGFP[LVA] and determining 1) what is the half-life in C. trachomatis, and 2) whether the short-lived GFP[LVA] reporter gene could accurately track and predict the transient mRNA profile of the early chlamydial gene, euo during infection.
Comments
Mentor: Dr. Li Shen M.D/PhD