Difference between revisions of "Part:BBa K3376000"
 
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<partinfo>BBa_K3376000 short</partinfo>
 
<partinfo>BBa_K3376000 short</partinfo>
  
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Lactate dehydrogenase catalyzes the fermentation of pyruvate to lactic acid. It plays a pivotal role in the glucose metabolic pathway connecting glycolysis, and is recognized as an essential gene and expressed at high level in S. mutans. Its promoter was cloned out from the gDNA of S. mutans and tested in transformed E. coli.  
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Lactate dehydrogenase catalyzes the fermentation of pyruvate to lactic acid. It plays a pivotal role in the glucose metabolic pathway connecting glycolysis, and is recognized as an essential gene and expressed at high level in S. mutans. Its ldhp promoter containing the presumed native RBS was cloned out from the gDNA of S. mutans and tested in transformed E. coli.  
 
   
 
   
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=== Expression ===
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The fluorescence expression levels were measured for lactate dehydrogenase promoter (ldhp) [ldhp-GFP-Tr/pSB1C3 (BBa_K3376002)] and thiol peroxidase promoter (tpxp) [tpxp-GFP-Tr/pSB1C3 (BBa_K3376004)] activity of S. mutans. GFP was detected at Ex/Em = 483/513. As shown in the figure, the expression level of ldhp-driven GFP was very high compared to a regulated promoter (tpxp) and a vector-only control.
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[[File:T--Mingdao--ww1.png|450px|center]]
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=== Transformation ===
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The ldhp-GFP-Tr/pSB1C3 [BBa_K3376002] was transformed into E. coli Nissle strain. The high expression of green fluorescent protein was observed under a blue led light.
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[[File:T--Mingdao--ww2.png|600px|left]]
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<br><br><br><br>
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<br><br><br><br>
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<br><br><br><br>
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<br><br><br><br>
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= CHARACTERIZATION by MINGDAO IGEM 2021 =
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In our project, we want to create phage reporter to detect pathogen through phages’ specific recognition. Therefore, we need a universal promoter to drive the reporter genes in different bacteria as many as possible. We chose the S. mutans lactate dehydrogenase promoter (ldhp) which activity was demonstrated in E. coli in our iGEM 2020 project and in many Gram-positive bacteria in research papers. The ldhp promoters were assembled on pTol2 plasmid backbone ([[Part:BBa_K3728002]]) with GFP ([[Part:BBa_K3728005]]), RFP ([[Part:BBa_K3728006]]) genes followed by a double terminator. The standard BBa_J04450 (lac promoter-driven RFP Coding Device) ([[Part:BBa_K3728003]]) was also assembled on pTol2 vector as a control. The ldhp promoter activities were characterized by In vitro transcription-translation (TXTL) assay, compared to lac promoter (lacp), and in Salmonella typhimurium strain LT2 (our phage typing target).
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<br>
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=== Characterization I – TXTL assay ===
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In vitro transcription-translation (TXTL) approach benefits synthetic biology in cell-free system <sup>1,2</sup>. We used it to characterize promoter activity in vitro and to drive phage machinery to package its own genome. The data in Fig. 1 showed ldhp promoter can drive high GFP expression in TXTL prepared using E. coli Rosetta 2(DE3) strains as bacterial lysates.
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[[File:T--Mingdao--Contribution Fig1-1.png|500px|left]]
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<br><br><br><br><br><br><br><br>
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Fig. 1. GFP fluorescence intensities were detected in TXTL and measured at Ex/Em=488/530nm for KanR/pTol2([[Part:BBa_K3728004]]) (Control) and ldhp-GFP-Tr/pTol2 ([[Part:BBa_K3728005]]).
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<br><br><br><br><br><br>
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=== Characterization II – compared to Lac promoter ===
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As expected, Fig. 2 represented high RFP intensities of ldhp-RFP in TXTL. And the activity of ldhp was much stronger than that of lacp because E. coli Rosetta 2 (DE3)-based TXTL system contains lac repressor negatively controlling lac promoter<sup>3</sup>. The enhanced promoter activities were detected in the transformed E. coli DH5α with the same plasmids, where lacp was much higher than ldhp (Fig. 3).
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[[File:T--Mingdao--Contribution Fig2-1.png|500px|left]]
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<br><br><br><br><br><br><br><br>
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Fig. 2. RFP fluorescence intensities were detected in TXTL and measured at Ex/Em=586/611nm for KanR/pTol2 (Control) ([[Part:BBa_K3728004]]), ldhp-RFP-Tr/pTol2 ([[Part:BBa_K3728006]]) and J04450/pTol2 (lacp-RFP) ([[Part:BBa_K3728003]])
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[[File:T--Mingdao--Contribution Fig3-1.png|500px|left]]
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<br><br><br><br><br><br><br><br><br><br>
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Fig. 3. RFP fluorescence intensities at Ex/Em=586/611nm were detected in the transformed E. coli with the plasmids of KanR/pTol2 (Control) ([[Part:BBa_K3728004]]), ldhp-RFP-Tr/pTol2 ([[Part:BBa_K3728006]]) and J04450/pTol2 (lacp-RFP) ([[Part:BBa_K3728003]]). The inset photos were pictured under visible light.
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<br><br><br><br>
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=== Characterization III – expression in Salmonella typhimurium strain LT2 ===
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ldhp-RFP was assembled on pBR322-based pSB6C1 ([[Part:BBa_K3728013]]). The resulting vector is able to transform Salmonella typhimurium LT2 with the appearance of red colonies on LB agar plate with 25 µg/mL of chloramphenicol (Fig. 4), showing the ldhp activity in the Gram-negative Salmonella cells.
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<br>
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[[File:T--Mingdao--Contribution Fig4-1.jpeg|300px|left]]
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<br><br><br><br><br><br><br><br>
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Fig. 4. Red colonies of the transformed E. coli with ldhp-RFP/pSB6C1 ([[Part:BBa_K3728013]]) plasmid formed on a Cm LB agar plate.
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<br>
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=== Reference ===
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# Garenne D, Noireaux V. Cell-free transcription-translation: engineering biology from the nanometer to the millimeter scale. Curr Opin Biotechnol. 2019 Aug;58:19-27. doi: 10.1016/j.copbio.2018.10.007.
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# Rustad M, Eastlund A, Marshall R, Jardine P, Noireaux V. Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System. J Vis Exp. 2017 Aug 17;(126):56144. doi: 10.3791/56144.
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# Rosetta™ 2(DE3) pLacI Competent Cells – Novagen (Sigma-Aldrich)
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===<span class='h3bb'>Sequence and Features</span>===
 
===<span class='h3bb'>Sequence and Features</span>===
 
<partinfo>BBa_K3376000 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3376000 SequenceAndFeatures</partinfo>

Latest revision as of 23:49, 16 September 2021


S. mutans lactate dehydrogenase promoter (ldhp)

Lactate dehydrogenase catalyzes the fermentation of pyruvate to lactic acid. It plays a pivotal role in the glucose metabolic pathway connecting glycolysis, and is recognized as an essential gene and expressed at high level in S. mutans. Its ldhp promoter containing the presumed native RBS was cloned out from the gDNA of S. mutans and tested in transformed E. coli.

Expression

The fluorescence expression levels were measured for lactate dehydrogenase promoter (ldhp) [ldhp-GFP-Tr/pSB1C3 (BBa_K3376002)] and thiol peroxidase promoter (tpxp) [tpxp-GFP-Tr/pSB1C3 (BBa_K3376004)] activity of S. mutans. GFP was detected at Ex/Em = 483/513. As shown in the figure, the expression level of ldhp-driven GFP was very high compared to a regulated promoter (tpxp) and a vector-only control.

T--Mingdao--ww1.png

Transformation

The ldhp-GFP-Tr/pSB1C3 [BBa_K3376002] was transformed into E. coli Nissle strain. The high expression of green fluorescent protein was observed under a blue led light.

T--Mingdao--ww2.png


















CHARACTERIZATION by MINGDAO IGEM 2021

In our project, we want to create phage reporter to detect pathogen through phages’ specific recognition. Therefore, we need a universal promoter to drive the reporter genes in different bacteria as many as possible. We chose the S. mutans lactate dehydrogenase promoter (ldhp) which activity was demonstrated in E. coli in our iGEM 2020 project and in many Gram-positive bacteria in research papers. The ldhp promoters were assembled on pTol2 plasmid backbone (Part:BBa_K3728002) with GFP (Part:BBa_K3728005), RFP (Part:BBa_K3728006) genes followed by a double terminator. The standard BBa_J04450 (lac promoter-driven RFP Coding Device) (Part:BBa_K3728003) was also assembled on pTol2 vector as a control. The ldhp promoter activities were characterized by In vitro transcription-translation (TXTL) assay, compared to lac promoter (lacp), and in Salmonella typhimurium strain LT2 (our phage typing target).


Characterization I – TXTL assay

In vitro transcription-translation (TXTL) approach benefits synthetic biology in cell-free system 1,2. We used it to characterize promoter activity in vitro and to drive phage machinery to package its own genome. The data in Fig. 1 showed ldhp promoter can drive high GFP expression in TXTL prepared using E. coli Rosetta 2(DE3) strains as bacterial lysates.


T--Mingdao--Contribution Fig1-1.png









Fig. 1. GFP fluorescence intensities were detected in TXTL and measured at Ex/Em=488/530nm for KanR/pTol2(Part:BBa_K3728004) (Control) and ldhp-GFP-Tr/pTol2 (Part:BBa_K3728005).








Characterization II – compared to Lac promoter

As expected, Fig. 2 represented high RFP intensities of ldhp-RFP in TXTL. And the activity of ldhp was much stronger than that of lacp because E. coli Rosetta 2 (DE3)-based TXTL system contains lac repressor negatively controlling lac promoter3. The enhanced promoter activities were detected in the transformed E. coli DH5α with the same plasmids, where lacp was much higher than ldhp (Fig. 3).


T--Mingdao--Contribution Fig2-1.png









Fig. 2. RFP fluorescence intensities were detected in TXTL and measured at Ex/Em=586/611nm for KanR/pTol2 (Control) (Part:BBa_K3728004), ldhp-RFP-Tr/pTol2 (Part:BBa_K3728006) and J04450/pTol2 (lacp-RFP) (Part:BBa_K3728003)


T--Mingdao--Contribution Fig3-1.png











Fig. 3. RFP fluorescence intensities at Ex/Em=586/611nm were detected in the transformed E. coli with the plasmids of KanR/pTol2 (Control) (Part:BBa_K3728004), ldhp-RFP-Tr/pTol2 (Part:BBa_K3728006) and J04450/pTol2 (lacp-RFP) (Part:BBa_K3728003). The inset photos were pictured under visible light.






Characterization III – expression in Salmonella typhimurium strain LT2

ldhp-RFP was assembled on pBR322-based pSB6C1 (Part:BBa_K3728013). The resulting vector is able to transform Salmonella typhimurium LT2 with the appearance of red colonies on LB agar plate with 25 µg/mL of chloramphenicol (Fig. 4), showing the ldhp activity in the Gram-negative Salmonella cells.


T--Mingdao--Contribution Fig4-1.jpeg









Fig. 4. Red colonies of the transformed E. coli with ldhp-RFP/pSB6C1 (Part:BBa_K3728013) plasmid formed on a Cm LB agar plate.




Reference

  1. Garenne D, Noireaux V. Cell-free transcription-translation: engineering biology from the nanometer to the millimeter scale. Curr Opin Biotechnol. 2019 Aug;58:19-27. doi: 10.1016/j.copbio.2018.10.007.
  2. Rustad M, Eastlund A, Marshall R, Jardine P, Noireaux V. Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System. J Vis Exp. 2017 Aug 17;(126):56144. doi: 10.3791/56144.
  3. Rosetta™ 2(DE3) pLacI Competent Cells – Novagen (Sigma-Aldrich)




Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]