Team:NYMU-Taipei/Experiments/Functional Assays

From 2013.igem.org

(Difference between revisions)

Latest revision as of 04:20, 28 September 2013

National Yang Ming University

Notes

In this page, we list all the functional assays that we have done with summarized results.

For combined and organized sets of our 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
Items: E0840, J23101+E0840,R0065+E0840, pLac+E0840
Testing: OD600, fluorescence 490/530, 485/525 and 470/530 in ELISA plate reader
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
Items: 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

Purpose: Measuring TrxCp+E0840 with respect to different H₂O₂ concentrations
Scale: 8 short terms (2ml), sample retrieved at 4 hours
Items: 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

Purpose: Measuring Hemhp+E0840 with respect to different H₂O₂ concentrations
Scale: 10 short terms (2 ml), sample retrieve at 4 hours
Items: 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 pSB1C3 backbone

2013-08-27

Purpose: Measuring sufAp(K554003)+E0840 with respect to different H₂O₂ concentrations
Scale: 10 short terms (2 ml), sample retrieve at 4 hours
Items: 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

Items: E0840, J23101+E0840, J23102+E0840, R0082+E0840
Scale: 10 short terms (2 ml), sample retrieve at 4 hours
Results: When J23101 is standardized as 1, the strength of J23102 is 1.12, and R0082 is 1.25. Since R0082 is positively regulated, R0082 is too leaky so we chose not to use it anymore.

2013-08-31

Purpose: Measuring 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, .
Items: E0840, J23101+E0840, J23104+E0840, J23119+E0840, K592006+ E0840
Results:
1. 40 replicates of J23101 receive a R² value 0.9757 .
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.

2013-09-05

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.
Items: J23101

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.
Items: 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) on pSB1C3 backbone.

2013-09-20

Purpose: Knowing the reaction of our 3 new AhpCp1000 derived OxyR-induced promoters to H₂O₂
Scale: 10 short terms (2ml) from 3 to 7.5 hours, samples retrieved every 30 minutes.
Items: AhpCp1,AhpCpd1 and AhpCp2; each with three different H₂O₂ concentrations(OmM, 0.1mM, 1mM)on pSB1C3 backbone.

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.
Scale: 10 short terms (2ml) from 3 to 7.5 hours, samples retrieved every 30 minutes.
Items: AhpCp1+E0840, AhpCp2+E0840, sufAp+E0840, R0065+E0840,J23101+E0840, TrxC+E0840. The last two promotors aree treated with 8 different H₂O₂ concentrations on pSB1C3 backbone.
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.
Items: AhpCp1+E0840, AhpCp2+E0840, TrxCp+E0840; each promoter of the three with 6 different H₂O₂ concentration (0mM, 0.01mM, 0.05mM, 0.1mM, 0.5mM, 1mM) on pSB1C3 backbone

2013-09-26 - 2

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

on pSB1C3 backbone

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

@@ Line 1: / Line 1: @@
 {{:Team:NYMU-Taipei/Header}}
-This page is a list of all the functional assays that we have done with simple results.
+=Notes=
+In this page, we list all the functional assays that we have done with summarized results.
-For a more combined and organized set of results, see our [[Team:NYMU-Taipei/Experiment/Wet_Lab | Data page]].
+For combined and organized sets of our results, see our [[Team:NYMU-Taipei/Experiment/Wet_Lab | Data page]].
 === 2013-07-29 ===
 * Scale: long term (25ml), samples retrieved from 2 to 6 hours once every 30 minutes.
-* Items: E0240,E0240+H<sub>2</sub>O<sub>2</sub>, pLac+E0840, pLac+I13507, pNHaA+E0240, pLac+I13507+H<sub>2</sub>O<sub>2</sub> on pSB1A2 backbone
+* Items: E0240, E0240+H<sub>2</sub>O<sub>2</sub>, pLac+E0840, pLac+I13507, pNHaA+E0240, pLac+I13507+H<sub>2</sub>O<sub>2</sub> 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
@@ Line 29: / Line 30: @@
 === 2013-08-08 ===
-*Purpose: Finding the impact of high temperature (44C) on the various promoters.
+* Purpose: Finding the impact of high temperature (44C) on the various promoters.
-*Scale: long term, short term
+* Scale: long term, short term
-*Item: E0840, J23101+E0840,R0065+E0840, pLac+E0840
+* Items: E0840, J23101+E0840,R0065+E0840, pLac+E0840
-*Testing: OD600, fluorescence 490/530, 485/525 and 470/530  in ELISA plate reader
+* 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:
 *# 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.
@@ Line 39: / Line 39: @@
 === 2013-08-14 ===
-*Scale: long term (25ml) for 8 hours
+* Scale: long term (25ml) for 8 hours
-*Item: E0840, J23101+E0840, pLac+E0840,HemHp+E0840 with 6 different H<sub>2</sub>O<sub>2</sub> concentrations (0mM, 0.01mM, 0.05mM, 0.1mM, 0.5mM, 1mM) on pSB1A2 backbone
+* Items: E0840, J23101+E0840, pLac+E0840,HemHp+E0840 with 6 different H<sub>2</sub>O<sub>2</sub> concentrations (0mM, 0.01mM, 0.05mM, 0.1mM, 0.5mM, 1mM) on pSB1A2 backbone
-*Testing: OD600 and fluorescence 485/525 in ELISA plate reader
+* Testing: OD600 and fluorescence 485/525 in ELISA plate reader
 === 2013-08-20 ===
-*Scale: 8 short terms (2ml), sample retrieved at 4 hours
+* Purpose: Measuring  TrxCp+E0840 with respect to different H<sub>2</sub>O<sub>2</sub> concentrations
-*Item: E0840, J23101+E0840, R0065+E0840, TrxC+E0840 with 7 different H<sub>2</sub>O<sub>2</sub> concentration (0mM, 0.01mM, 0.05mM, 0.1mM, 0.5mM, 1mM, 5mM) on pSB1A2 backbone
+* Scale: 8 short terms (2ml), sample retrieved at 4 hours
-*Testing: OD600 and fluorescence 485/525 in ELISA plate reader
+* Items: E0840, J23101+E0840, R0065+E0840, TrxC+E0840 with 7 different H<sub>2</sub>O<sub>2</sub> 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 H<sub>2</sub>O<sub>2</sub> concentration rises until it reach 1 mM, and the ''E. coli'' K-12 MG1655 dies at 5 mM.
+* Result: the strength of TrxC promotor enhance as H<sub>2</sub>O<sub>2</sub> 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
+* Purpose: Finding the photometric and fluorometric value of the medium as background
-*Item: M9 medium and PBS medium
+* Item: M9 medium and PBS medium
 === 2013-08-26 ===
-*Item: J23101+E0840, R0051+ E0840, TrxCp+E0840, Hemhp+E0840 with 7 different H<sub>2</sub>O<sub>2</sub> concentrations (0mM, 0.01mM, 0.05mM, 0.1mM, 0.5mM, 1mM, 5mM) on pSB1A2 backbone
+* Purpose: Measuring  Hemhp+E0840 with respect to different H<sub>2</sub>O<sub>2</sub> concentrations
+* Scale: 10 short terms (2 ml), sample retrieve at 4 hours
+* Items: J23101+E0840, R0051+ E0840, TrxCp+E0840, Hemhp+E0840 with 7 different H<sub>2</sub>O<sub>2</sub> concentrations (0mM, 0.01mM, 0.05mM, 0.1mM, 0.5mM, 1mM, 5mM) on pSB1C3 backbone
 === 2013-08-27 ===
-*Purpose: Finding the reaction SufAp(K554003)+E0840 to H<sub>2</sub>O<sub>2</sub>
+* Purpose:  Measuring  sufAp(K554003)+E0840 with respect to different H<sub>2</sub>O<sub>2</sub> concentrations
-*Scale: 10 short terms (2 ml), sample retrieve at 4 hours
+* Scale: 10 short terms (2 ml), sample retrieve at 4 hours
-*Item: J23101+E0840, R0040+E0840, R0074+E0840, SufAp+E0840with 7 different H<sub>2</sub>O<sub>2</sub> concentrations (0mM, 0.01mM, 0.05mM, 0.1mM, 0.5mM, 1mM, 5mM) on pSB1A2 backbone
+* Items: J23101+E0840, R0040+E0840, R0074+E0840, sufAp+E0840with 7 different H<sub>2</sub>O<sub>2</sub> 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
+* Items: 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
+* Scale: 10 short terms (2 ml), sample retrieve at 4 hours
-* 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.
+* Results: When J23101 is standardized as 1, the strength of J23102 is 1.12, and R0082 is 1.25. Since R0082 is positively regulated, R0082 is too leaky so we chose not to use it anymore.
 === 2013-08-31 ===
-* E0840, J23101 (replicates retrieved backwards from samples 10 till 1, thus the first time point only grew for 2hrs)
+* Purpose: Measuring 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.
+* Items: E0840, J23101+E0840, J23104+E0840, J23119+E0840, K592006+ E0840
+* Results:
-=== 2013-09-02 ===
+*#40 replicates of J23101 receive a R<sup>2</sup> value 0.9757 .
-*
+*#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.
 === 2013-09-05 ===
-* Purpose: Measuring NTU_Taida's parts.[[https://2013.igem.org/Team:NYMU-Taipei/HumanPractice/HumCollab  |Human Practice Collaboration]]
+* Purpose: Measuring NTU_Taida's parts.[[https://2013.igem.org/Team:NYMU-Taipei/HumanPractice/HumCollab Human Practice Collaboration]]
 === 2013-09-07 ===
-* Purpose: Measure J23101 under different H<sub>2</sub>O<sub>2</sub> concentrations.
+* Purpose: Since J23101 is taken as the standard in the reporting assay experiments, we measure the reaction of J23101 under different H<sub>2</sub>O<sub>2</sub> concentrations.
-* Time points retrieved in the order 7 to 1, then 10 to 8.
+* Scale: 10 shorts terms, time points retrieving in the order 7 to 1, then 10 to 8.
+* Items: J23101
 === 2013-09-10 ===
-* J23101
+* 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.
+* Items: E0840, J23101+ E0840 with 6 different H<sub>2</sub>O<sub>2</sub> concentration (0mM, 0.05mM, 0.1mM, 0.5mM, 1mM, 2mM)
 === 2013-09-19 ===
-* Measured TrxC promoter with respect to different H<sub>2</sub>O<sub>2</sub> concentrations.
+* Purpose: Measuring TrxCp+E0840 with respect to different H<sub>2</sub>O<sub>2</sub> 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 H<sub>2</sub>O<sub>2</sub> concentrations (0mM, 0.01mM, 0.05mM, 0.1mM, 0.5mM, 1mM) on pSB1C3 backbone.
 === 2013-09-20 ===
 * Purpose: Knowing the reaction of our 3 new AhpCp1000 derived OxyR-induced promoters to H<sub>2</sub>O<sub>2</sub>
-* Item: AhpCp1,AhpCpd1 and AhpCp2; each with three different H<sub>2</sub>O<sub>2</sub> concentrations(OmM, 0.1mM, 1mM)
+* Scale: 10 short terms (2ml) from 3 to 7.5 hours, samples retrieved every 30 minutes.
+* Items: AhpCp1,AhpCpd1 and AhpCp2; each with three different H<sub>2</sub>O<sub>2</sub> concentrations(OmM, 0.1mM, 1mM)on pSB1C3 backbone.
 === 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 H<sub>2</sub>O<sub>2</sub> concentrations.
+* Scale: 10 short terms (2ml) from 3 to 7.5 hours, samples retrieved every 30 minutes.
+* Items: AhpCp1+E0840, AhpCp2+E0840, sufAp+E0840, R0065+E0840,J23101+E0840, TrxC+E0840. The last two promotors aree treated with 8 different H<sub>2</sub>O<sub>2</sub> concentrations on pSB1C3 backbone.
 * Results:The M9 minimal medium has the possibility to interferes with the H<sub>2</sub>O<sub>2</sub>, 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.
+* 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 H<sub>2</sub>O<sub>2</sub> concentration (0mM, 0.01mM, 0.05mM, 0.1mM, 0.5mM, 1mM)
+* Items: AhpCp1+E0840, AhpCp2+E0840, TrxCp+E0840; each promoter of the three with 6 different H<sub>2</sub>O<sub>2</sub> concentration (0mM, 0.01mM, 0.05mM, 0.1mM, 0.5mM, 1mM) on pSB1C3 backbone
 === 2013-09-26 - 2 ===
-*Scale: 10 short terms (2ml) from 3 to 7.5 hours, samples retrieved every 30 minutes.
+* 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 H<sub>2</sub>O<sub>2</sub> concentration (0mM, 0.01mM, 0.05mM, 0.1mM, 0.5mM, 1mM)
+* Items: AhpCp1000+E0840, AhpCp2D1+E0840; both with 6 different H<sub>2</sub>O<sub>2</sub> concentration (0mM, 0.01mM, 0.05mM, 0.1mM, 0.5mM, 1mM)
+on pSB1C3 backbone
 {{:Team:NYMU-Taipei/Footer}}