Difference between revisions of "Part:BBa J23105"
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[[File:T--GreatBay_China--cons.png|800px]]
 
[[File:T--GreatBay_China--cons.png|800px]]
 
<p>The result indicate that the strength of J23119, J23105, and J23101 are about the same as described by team iGEM2006_Berkeley, and the fluorescence increases as the copy number of the vector increases</p>
 
<p>The result indicate that the strength of J23119, J23105, and J23101 are about the same as described by team iGEM2006_Berkeley, and the fluorescence increases as the copy number of the vector increases</p>
 +
 +
===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>
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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="527"
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        height="366">
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  </body>
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</html>

Revision as of 21:55, 28 September 2019

constitutive promoter family member

BerkiGEM2006-PromotersEppendorfs.jpg
BerkiGEM2006-Promoters.jpg

 
 Variant RFP (au)
 J23112           1
 J23103           17
 J23113           21
 J23109           106
 J23117           162
 J23114           256
 J23115           387
 J23116           396
 J23105           623
 J23110           844
 J23107           908
 J23106           1185
 J23108           1303
 J23118           1429
 J23111           1487
 J23101           1791
 J23104           1831
 J23102           2179
 J23100           2547
PBca1020-r0040.jpg

Constitutive promoter family
Parts J23100 through J23119 are a family of constitutive promoter parts isolated from a small combinatorial library. J23119 is the "consensus" promoter sequence and the strongest member of the family. All parts except J23119 are present in plasmid J61002. Part J23119 is present in pSB1A2. This places the RFP downstream of the promoter. Reported activities of the promoters are given as the relative fluorescence of these plasmids in strain TG1 grown in LB media to saturation. See part BBa_J61002 for details on their use.

These promoter parts can be used to tune the expression level of constitutively expressed parts. The NheI and AvrII restriction sites present within these promoter parts make them a scaffold for further modification. JCAraw




Manchester 2017 used this part to create part LowPromoter_PduD(1-20)_mCherry (BBa_K2213006). This promoter was combined with PduD(1-20) to create a tag with lower expression levels. The mCherry tagged PduD(1-20) localisation tag displayed lower fluorescence levels under the low promoter as compared to under medium (BBa_K2213007) and high strength (BBa_K2213008) promoters, demonstrating correct function.

More information can be found here: https://parts.igem.org/Part:BBa_K2213006

TagExpression500p.jpg

GreatBay_China 2018:
Team GreatBay_China 2018 characterized J23119, Part:BBa_J23105, and Part:BBa_J23101 by assembling them with Part:BBa_B0034 and a sfGFPPart:BBa_I746916 on three vectors: pUC20 (copy number about 500/cell), pR6K (copy number about 15/cell), pSC101 (copy number about 2/cell). Then we measured the fluorescence by Flow Cytometry as a reference for the TALE stabilized promoter library.
T--GreatBay China--cons.png

The result indicate that the strength of J23119, J23105, and J23101 are about the same as described by team iGEM2006_Berkeley, and the fluorescence increases as the copy number of the vector increases

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