Team:NYMU-Taipei/Experiments/Functional Assays

From 2013.igem.org

Revision as of 20:38, 27 September 2013

This page creates lists the functional assays that we have done.

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
• Result: 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:
  • MG1655 dies above concentrations of 5mM H₂O₂
  • 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
• Result:
  1. Achieve maximum after 6 hours??
  2. 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.
  3. We finally settled on using the wavelength 485/525 for measuring GFP fluorescence.

20130814

• 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

20130820

• Scale: 8 short terms (4ml), 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.

20130825

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

20130826

• 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

20130827

20130830

• 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.

20130831

• E0840, J23101 (replicates retrieved backwards from samples 10 till 1, thus the first time point only grew for 2hrs)
• R²=0.9757 for 40 replicates.
• J23119 J23104 K592006 are almost the same as background, thus we are doubting that it is incorrect.

20130902

20130905

• Purpose: Measuring NTU_Taida's parts.

20130907

• Purpose: Measure J23101 under different H₂O₂ concentrations.
• Time points retrieved in the order 7 to 1, then 10 to 8.

20130910

• J23101

20130919

• Measured TrxC promoter with respect to different H₂O₂ concentrations.

20130920

• Measured AhpCp1 AhpCpd1 AhpCp2

20130925

• Tried M9 medium with different H₂O₂ concentrations. Found out that M9 probably interferes with the H₂O₂.

20130926 - 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)

20130926 - 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)