Team:NYMU-Taipei/Experiments/Functional Assays

From 2013.igem.org

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Revision as of 23:59, 27 September 2013

National Yang Ming University

This page is a list of all the functional assays that we have done with simple results.

For a more combined and organized set of results, see our Data page.

2013-07-29

Scale: long term (25ml), samples retrieved from 2 to 6 hours once every 30 minutes.
Items: E0240,E0240+H₂O₂, pLac+E0840, pLac+I13507, pNHaA+E0240, pLac+I13507+H₂O₂ on pSB1A2 backbone
medium: M9 minimal medium
Measuring: OD600 measured in a cuvette via spectrometer, fluorescence 485/525 (GFP) and 584/607 (RFP) in ELISA plate reader
Results: the strength of NHaA promotor is so weak that we decided to switch our safety biobrick to the next design.

2013-08-02

Purpose: Finding the suitable range of H₂O₂ concentration to test our promoters.
Items: pLac+E0840 with 8 different H₂O₂ concentrations (OmM, 0.05mM, 0.1mM, 0.5mM, 1mM, 5mM, 10mM, 50mM) on pSB1A2 backbone
Testing: Both OD600 and fluorescence 485/525 (GFP) both measured using the ELISA plate reader.
Results:
1. MG1655 dies above concentrations of 5mM H₂O₂
2. The data measured before 3 hours seem less reliable.

2013-08-04

Purpose: Measure the basal expressions of various promoters using smaller volume but more replicates.
Scale: 1 long term (25ml) samples retrieved at 0, 4, 6, 8, 10, 12 hours, and 7 short terms (4ml) retrieved at 0 and 4 hours.
Items: E0840, R0065+E0840, pLac+E0840, pNHaA+E0840, J23101+E0840, I13507, R0065+I13507, pLac+I13507, pNHaA+E0840, J23101+I13507 all on pSB1A2 backbone
Testing: OD600 and fluorescence 490/530, 485/525 and 470/530 (all GFP) in ELISA plate reader
Results: only R0065+E0840 ,J23101+E0840, J23101+I13507, pLac+E0840, pLac+I13507 show efficient function. This is an efficient way for creating biological replicates.

2013-08-08

Purpose: Finding the impact of high temperature (44C) on the various promoters.
Scale: long term, short term
Item: E0840, J23101+E0840,R0065+E0840, pLac+E0840
Testing: OD600, fluorescence 490/530, 485/525 and 470/530 in ELISA plate reader
first temperature testing--suggest that plac is repressed under 44C, R0065 as well
Results:
1. The pLac promoter is highly repressed under 44C compared to at 37C, and R0065 has relatively small repression. As a result, we bagan searching for another repressible promotor in place of pLac.
2. We finally settled on using the wavelength 485/525 for measuring GFP fluorescence.

2013-08-14

Scale: long term (25ml) for 8 hours
Item: E0840, J23101+E0840, pLac+E0840,HemHp+E0840 with 6 different H₂O₂ concentrations (0mM, 0.01mM, 0.05mM, 0.1mM, 0.5mM, 1mM) on pSB1A2 backbone
Testing: OD600 and fluorescence 485/525 in ELISA plate reader

2013-08-20

Scale: 8 short terms (2ml), sample retrieved at 4 hours
Item: E0840, J23101+E0840, R0065+E0840, TrxC+E0840 with 7 different H₂O₂ concentration (0mM, 0.01mM, 0.05mM, 0.1mM, 0.5mM, 1mM, 5mM) on pSB1A2 backbone
Testing: OD600 and fluorescence 485/525 in ELISA plate reader
We started washing with PBS instead of M9 medium.
Result: the strength of TrxC promotor enhance as H₂O₂ concentration rises until it reach 1 mM, and the E. coli K-12 MG1655 dies at 5 mM.

2013-08-25

Purpose: Finding the photometric and fluorometric value of the medium as background
Item: M9 medium and PBS medium

2013-08-26

Item: J23101+E0840, R0051+ E0840, TrxCp+E0840, Hemhp+E0840 with 7 different H₂O₂ concentrations (0mM, 0.01mM, 0.05mM, 0.1mM, 0.5mM, 1mM, 5mM) on pSB1A2 backbone

2013-08-27

Purpose: Finding the reaction SufAp(K554003)+E0840 to H₂O₂
Scale: 10 short terms (2 ml), sample retrieve at 4 hours
Item: J23101+E0840, R0040+E0840, R0074+E0840, SufAp+E0840with 7 different H₂O₂ concentrations (0mM, 0.01mM, 0.05mM, 0.1mM, 0.5mM, 1mM, 5mM) on pSB1A2 backbone

2013-08-30

Item: E0840, J23101+E0840, J23102+E0840, R0082+E0840
Result: When J23101 is standardized as 1, the strength of J23102 is 1.12, and R0082 is 1.25. Since R0082 is positively regulated
J23102 is 1.12x stronger than J23101, R0082 is 1.25x stronger than J23101. R0082 is too leaky so we chose not to use it anymore.

2013-08-31

Purpose: Measure the strength of different constitutive promotors
Scale: 10 short terms. Replicates retrieved backwards from samples 10 till 1, thus the first time point only grew for 2hrs. That is to say, samples are retrieved every 30 minutesfrom from 2 to 6.5 hours, .
Item: E0840, J23101+E0840, J23104+E0840, J23119+E0840, K592006+ E0840
Results:
1. R²=0.9757 for 40 replicates of J23101.
2. J23119+E0840, J23104+E0840, and K592006+E0840 are almost the same as background, thus we are doubting that it is incorrect. Moreover, we select J23102 as the constitutive promoter in the circuit.

2013-09-02

2013-09-05

Purpose: Measuring NTU_Taida's parts.[Human Practice Collaboration]

2013-09-07

Purpose: Since J23101 is taken as the standard in the reporting assay experiments, we measure the reaction of J23101 under different H₂O₂ concentrations.
Scale: 10 shorts terms, time points retrieving in the order 7 to 1, then 10 to 8.

2013-09-10

Purpose: Creating more duplicates of J23101 to standardized is value
Scale: 10 short terms (2ml) from 3 to 7.5 hours, samples retrieved every 30 minutes.
Item: E0840, J23101+ E0840 with 6 different H₂O₂ concentration (0mM, 0.05mM, 0.1mM, 0.5mM, 1mM, 2mM)

2013-09-19

Purpose: Measuring TrxCp+E0840 with respect to different H₂O₂ concentrations.
Scale: 10 shorts terms, time points retrieving in the order 1 to 2, then 10 to 3.
Item: E0840, J23101+E0840, TrxCp+E0840 with 6 different H₂O₂ concentrations (0mM, 0.01mM, 0.05mM, 0.1mM, 0.5mM, 1mM)

2013-09-20

Purpose: Knowing the reaction of our 3 new AhpCp1000 derived OxyR-induced promoters to H₂O₂
Item: AhpCp1,AhpCpd1 and AhpCp2; each with three different H₂O₂ concentrations(OmM, 0.1mM, 1mM)

2013-09-25

Purpose: To simplify our reporting assay protocols, we change our medium into M9 minimal medium, the E. coli K-12 MG1655 is cultivaed directly in 96-well plates.
Item: AhpCp1+E0840, AhpCp2+E0840, SufAp+E0840, R0065+E0840,J23101+E0840, TrxC+E0840. The last two promotors aree treated with 8 different H₂O₂ concentrations.
Results:The M9 minimal medium has the possibility to interferes with the H₂O₂, so we restore to our original protocols.

2013-09-26 - 1

Scale: 10 short terms (2ml) from 3 to 7.5 hours, samples retrieved every 30 minutes.
Item: AhpCp1+E0840,AhpC2+E0840, TrxC+E0840; each promoter of the three with 6 different H₂O₂ concentration (0mM, 0.01mM, 0.05mM, 0.1mM, 0.5mM, 1mM)

2013-09-26 - 2

Scale: 10 short terms (2ml) from 3 to 7.5 hours, samples retrieved every 30 minutes.
Item: AhpCp1000+E0840, AhpCp2D1+E0840; both with 6 different H₂O₂ concentration (0mM, 0.01mM, 0.05mM, 0.1mM, 0.5mM, 1mM)

Retrieved from "http://2013.igem.org/Team:NYMU-Taipei/Experiments/Functional_Assays"

@@ Line 71: / Line 71: @@
 === 2013-08-31 ===
-* E0840, J23101 (replicates retrieved backwards from samples 10 till 1, thus the first time point only grew for 2hrs)
+* Purpose: Measure the strength of different constitutive promotors
-* R<sup>2</sup>=0.9757 for 40 replicates.
+* Scale: 10 short terms. Replicates retrieved backwards from samples 10 till 1, thus the first time point only grew for 2hrs. That is to say, samples are retrieved every 30 minutesfrom from 2 to 6.5 hours, .
-* J23119 J23104 K592006 are almost the same as background, thus we are doubting that it is incorrect.
+* Item: E0840, J23101+E0840, J23104+E0840, J23119+E0840, K592006+ E0840
+* Results:
+*#R<sup>2</sup>=0.9757 for 40 replicates of J23101.
+*#J23119+E0840, J23104+E0840, and K592006+E0840 are almost the same as background, thus we are doubting that it is incorrect. Moreover, we select J23102 as the constitutive promoter in the circuit.
 === 2013-09-02 ===