Team:NYMU-Taipei/Experiments/Functional Assays

From 2013.igem.org

National Yang Ming University


Contents

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

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"