Difference between revisions of "Part:BBa K2973020:Experience"

(→‎Experimental Design)
(→‎Thessaly 2019 Characterization)
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===Thessaly 2019 Characterization===
 
===Thessaly 2019 Characterization===
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Thessaly 2019 sought to characterize the coding sequence of TEM-optimized beta-lactamase (BBa_I757010) under the regulation of the constituve Anderson Family promoters BBa_J23100, BBa_J23105, BBa_J23106, BBa_J23119. To achieve that, the coding sequence was assembled with each promoter, a universal RBS (BBa_B0034) and a double terminator(BBa_B0015).  
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Thessaly 2019 sought to characterize the coding sequence of TEM-optimized beta-lactamase (BBa_I757010) under the regulation of the constituve Anderson Family promoters BBa_J23100, BBa_J23105, BBa_J23106, BBa_J23119. Beta-lactamase is an enzyme that hydrolyses beta-lactams (e.g. ampicillin) and is naturally found in procaryotic cells. A colorimetric assay has been developed using nitrocefin as a substrate which after hydrolysis from beta-lactamase changes the reaction color, from yellow (380nm) to red (490nm).
 +
 
 +
To achieve that, the coding sequence was assembled with each promoter, a universal RBS (BBa_B0034) and a double terminator(BBa_B0015). The parts were cloned in pSB1C3 and pSB1K3 and transformed into E. coli DH5a competent cells. For protein expression, the plasmids were transformed into E. coli BL21 (DE3) competent cells.
 +
 
 +
 
 +
For the beta-lactamase assay, we set up the following experimental design:
 +
 
 +
1. Grow BL21 (DE3) cells overnight in 5ml LB (~16h) at a shaken incubator, 37 degrees C / 210rpm
 +
2. The following morning, measure the OD600 of overnight cultures
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3. Dilute all cultures to OD600¬ = 0.05 in M9 minimal medium
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4. Grow cells 37 degrees C /210 RPM until OD600=0.4-0.6 (~2h)
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5. Dilute all cells to the same OD600 (e.g. 0.4)
 +
6. Load 160 of culture in a 96-well plate (do triplicates). Add 40 ul 0.5 uM nitrocefin for a final concentration of 100nM
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7. Measure the absorbance at 490nm (for nitrocefin hydrolysis) and 600nm (for cell growth) every 30 seconds for 25 minutes in a microplate reader. Shake between measurements.
 +
 
 +
To ensure that the absorbance shown corresponds only to enzymatic activity by beta-lactamase, we included 3 controls in the experiment.
 +
The first control has M9 medium only (no cells) and nitrocefin, the second has empty BL21 (DE3) cells (no plasmid) and nitrocefin, while the third has BL21 (DE3) cells containing the plasmid but not the part (empty plasmid).
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To obtain comparable results, we normalized all values by dividing OD490 by OD600.
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The results are shown in the graph below
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<html>
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  <head>
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      <title>HTML img Tag</title>
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  </head>
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  <body>
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      <img src="https://static.igem.org/mediawiki/parts/d/d5/Thessaly_2019_Characterization_Graph_Lactamase.jpeg" width="2262"
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        height="917">
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  </body>
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</html>
  
 
===User Reviews===
 
===User Reviews===

Revision as of 21:32, 28 September 2019


This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K2973020

Thessaly 2019 Characterization

Thessaly 2019 sought to characterize the coding sequence of TEM-optimized beta-lactamase (BBa_I757010) under the regulation of the constituve Anderson Family promoters BBa_J23100, BBa_J23105, BBa_J23106, BBa_J23119. Beta-lactamase is an enzyme that hydrolyses beta-lactams (e.g. ampicillin) and is naturally found in procaryotic cells. A colorimetric assay has been developed using nitrocefin as a substrate which after hydrolysis from beta-lactamase changes the reaction color, from yellow (380nm) to red (490nm).

To achieve that, the coding sequence was assembled with each promoter, a universal RBS (BBa_B0034) and a double terminator(BBa_B0015). The parts were cloned in pSB1C3 and pSB1K3 and transformed into E. coli DH5a competent cells. For protein expression, the plasmids were transformed into E. coli BL21 (DE3) competent cells.


For the beta-lactamase assay, we set up the following experimental design:

1. Grow BL21 (DE3) cells overnight in 5ml LB (~16h) at a shaken incubator, 37 degrees C / 210rpm 2. The following morning, measure the OD600 of overnight cultures 3. Dilute all cultures to OD600¬ = 0.05 in M9 minimal medium 4. Grow cells 37 degrees C /210 RPM until OD600=0.4-0.6 (~2h) 5. Dilute all cells to the same OD600 (e.g. 0.4) 6. Load 160 of culture in a 96-well plate (do triplicates). Add 40 ul 0.5 uM nitrocefin for a final concentration of 100nM 7. Measure the absorbance at 490nm (for nitrocefin hydrolysis) and 600nm (for cell growth) every 30 seconds for 25 minutes in a microplate reader. Shake between measurements.

To ensure that the absorbance shown corresponds only to enzymatic activity by beta-lactamase, we included 3 controls in the experiment. The first control has M9 medium only (no cells) and nitrocefin, the second has empty BL21 (DE3) cells (no plasmid) and nitrocefin, while the third has BL21 (DE3) cells containing the plasmid but not the part (empty plasmid). To obtain comparable results, we normalized all values by dividing OD490 by OD600.

The results are shown in the graph below

HTML img Tag

User Reviews

UNIQ2a0e2b468ecb402f-partinfo-00000001-QINU UNIQ2a0e2b468ecb402f-partinfo-00000002-QINU