Difference between revisions of "Part:BBa K3562002"
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===Experimental Plan===
 
===Experimental Plan===
 
Preparation of cell-free extracts: E. coli BL21 (DE3) are grown in 1 L of 2 x YTPG medium comprising 10 g/L yeast extract, 16 g/L tryptone, 5 g/L NaCl, 7 g/L K2HPO4, 3 g/L KH2PO4 and 18 g/L glucose in a 2.5-L shake flask at 37◦C and 250 rpm with inoculation of 10 mL overnight cultures. IPTG is added when OD600 reached 0.5 with a final concentration of 1 mM to induce T7 polymerase overexpression and target proteins pre-enrichment, and the cells are harvested by centrifugation at 5000 g for 15 min at 4◦C when OD600 was 3.0. Cell pellets are washed three times with ice-cold S30 buffer comprising 10 mM Tris-acetate (pH 8.2), 14 mM magnesium acetate, 60 mM potassium glutamate, and 2 mM dithiothreitol and then resuspended in 1 mL of S30 buffer per 1 g wet cells. The cells are disrupted via sonication or homogenization. The cell lysates are centrifuged at 30,000 g for 30 min at 4◦C to remove cell debris and insoluble components. Cell extracts are flash frozen in liquid nitrogen and stored at -80◦C until use.
 
Preparation of cell-free extracts: E. coli BL21 (DE3) are grown in 1 L of 2 x YTPG medium comprising 10 g/L yeast extract, 16 g/L tryptone, 5 g/L NaCl, 7 g/L K2HPO4, 3 g/L KH2PO4 and 18 g/L glucose in a 2.5-L shake flask at 37◦C and 250 rpm with inoculation of 10 mL overnight cultures. IPTG is added when OD600 reached 0.5 with a final concentration of 1 mM to induce T7 polymerase overexpression and target proteins pre-enrichment, and the cells are harvested by centrifugation at 5000 g for 15 min at 4◦C when OD600 was 3.0. Cell pellets are washed three times with ice-cold S30 buffer comprising 10 mM Tris-acetate (pH 8.2), 14 mM magnesium acetate, 60 mM potassium glutamate, and 2 mM dithiothreitol and then resuspended in 1 mL of S30 buffer per 1 g wet cells. The cells are disrupted via sonication or homogenization. The cell lysates are centrifuged at 30,000 g for 30 min at 4◦C to remove cell debris and insoluble components. Cell extracts are flash frozen in liquid nitrogen and stored at -80◦C until use.
 +
 
Cell-free protein synthesis: Standard CFPS reactions are performed at a volume of 16 μL in a 200 μL test-tube with incubation at 30 ◦C for 4 h. Each reaction mixture contains 4 μL of 4X premix solution comprising 4.8 mM ATP; 3.4 mM each of GTP, UTP, and CTP; 136.0 μg/mL L-5-formyl-5, 6, 7, 8-tetrahydrofolic acid; 680.0 μg/mL E. coli tRNA mixture; 520 mM potassium glutamate; 40 mM ammonium glutamate; 48 mM magnesium glutamate; 8 mM each of 20 amino acids; 1.32 mM nicotinamide adenine dinucleotide (NAD+); 1.08 mM coenzyme-A (CoA); 6 mM spermidine; 4 mM putrescine; 16 mM sodium oxalate; 132 mM phosphoenolpyruvate (PEP); 4 μL plasmid [final concentration was 15 μg/mL], 4 μL Milli-Q water and 4 μL cell extract (25% v/v).
 
Cell-free protein synthesis: Standard CFPS reactions are performed at a volume of 16 μL in a 200 μL test-tube with incubation at 30 ◦C for 4 h. Each reaction mixture contains 4 μL of 4X premix solution comprising 4.8 mM ATP; 3.4 mM each of GTP, UTP, and CTP; 136.0 μg/mL L-5-formyl-5, 6, 7, 8-tetrahydrofolic acid; 680.0 μg/mL E. coli tRNA mixture; 520 mM potassium glutamate; 40 mM ammonium glutamate; 48 mM magnesium glutamate; 8 mM each of 20 amino acids; 1.32 mM nicotinamide adenine dinucleotide (NAD+); 1.08 mM coenzyme-A (CoA); 6 mM spermidine; 4 mM putrescine; 16 mM sodium oxalate; 132 mM phosphoenolpyruvate (PEP); 4 μL plasmid [final concentration was 15 μg/mL], 4 μL Milli-Q water and 4 μL cell extract (25% v/v).
 +
 
Fluorescence detection: AHLs with different concentrations are added to the cell-free reactions. Microplate reader is used to detect and calculate the highest fluorescence outputs to indicate the concentration of AHLs, with LC-MS as the control.
 
Fluorescence detection: AHLs with different concentrations are added to the cell-free reactions. Microplate reader is used to detect and calculate the highest fluorescence outputs to indicate the concentration of AHLs, with LC-MS as the control.
  

Revision as of 13:56, 26 October 2020


pLasRV

las promoter from Pseudomonas aeruginosa

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
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 14


Usage and Biology

We found this well-characterized promoter pLasRV from P. aeruginosa cloned by Paul Freemont group in Imperial College, with in vivo and in vitro expression data thoroughly presented in the research articles [1]. Notably, researchers developed a cell-free biosensor based on this promoter to detect the concentration of OdDHL in sputum samples [2]. More attempts to work on this inducible promoter in iGEM will deepen the understanding of quorum sensing in P. aeruginosa and facilitate the exploitation of biomarker-based biosensors.

Figure 1 Characterization of inducible promoter pLasRV in cell-free systems and E. coli [1]
Figure 2 Overview of the LasRV cell-free biosensor [2]. A: graphical description of LasRV biosensor; B: fluorescence curve of 3OC12-HSL; C: specificity of LasRV cell-free biosensor to AHLs with different acyl chain lengths
Figure 3 Detection of sputum sample using the LasRV cell-free biosensor, with LC-MS as the control [2]


Experimental Plan

Preparation of cell-free extracts: E. coli BL21 (DE3) are grown in 1 L of 2 x YTPG medium comprising 10 g/L yeast extract, 16 g/L tryptone, 5 g/L NaCl, 7 g/L K2HPO4, 3 g/L KH2PO4 and 18 g/L glucose in a 2.5-L shake flask at 37◦C and 250 rpm with inoculation of 10 mL overnight cultures. IPTG is added when OD600 reached 0.5 with a final concentration of 1 mM to induce T7 polymerase overexpression and target proteins pre-enrichment, and the cells are harvested by centrifugation at 5000 g for 15 min at 4◦C when OD600 was 3.0. Cell pellets are washed three times with ice-cold S30 buffer comprising 10 mM Tris-acetate (pH 8.2), 14 mM magnesium acetate, 60 mM potassium glutamate, and 2 mM dithiothreitol and then resuspended in 1 mL of S30 buffer per 1 g wet cells. The cells are disrupted via sonication or homogenization. The cell lysates are centrifuged at 30,000 g for 30 min at 4◦C to remove cell debris and insoluble components. Cell extracts are flash frozen in liquid nitrogen and stored at -80◦C until use.

Cell-free protein synthesis: Standard CFPS reactions are performed at a volume of 16 μL in a 200 μL test-tube with incubation at 30 ◦C for 4 h. Each reaction mixture contains 4 μL of 4X premix solution comprising 4.8 mM ATP; 3.4 mM each of GTP, UTP, and CTP; 136.0 μg/mL L-5-formyl-5, 6, 7, 8-tetrahydrofolic acid; 680.0 μg/mL E. coli tRNA mixture; 520 mM potassium glutamate; 40 mM ammonium glutamate; 48 mM magnesium glutamate; 8 mM each of 20 amino acids; 1.32 mM nicotinamide adenine dinucleotide (NAD+); 1.08 mM coenzyme-A (CoA); 6 mM spermidine; 4 mM putrescine; 16 mM sodium oxalate; 132 mM phosphoenolpyruvate (PEP); 4 μL plasmid [final concentration was 15 μg/mL], 4 μL Milli-Q water and 4 μL cell extract (25% v/v).

Fluorescence detection: AHLs with different concentrations are added to the cell-free reactions. Microplate reader is used to detect and calculate the highest fluorescence outputs to indicate the concentration of AHLs, with LC-MS as the control.


Reference

[1]Chappel J et al., Validation of an entirely in vitro approach for rapid prototyping of DNA regulatory elements for synthetic biology. Nucleic Acids Research, 41 (2013): 3471–3481.
[2]Wen KY et al., A Cell-Free Biosensor for Detecting Quorum Sensing Molecules in P. aeruginosa-Infected Respiratory Samples. ACS Synth. Biol., 6 (2017): 2293-2301. [3]Zhang Y, Huang Q, Deng Z, Xu Y, Liu T. Enhancing the effificiency of cell-free protein synthesis system by systematic titration of transcription and translation components. Biochemical Engineering Journal, 138 (2018): 47-53.