Regulatory

Part:BBa_K1104204:Experience

Designed by: Ting-Yun Chiang   Group: iGEM13_NYMU-Taipei   (2013-09-16)
Revision as of 16:51, 27 October 2013 by S10101054 (Talk | contribs) (→‎Relative promoter strength testing)

Introduction

This part: AhpCp2D1 is one of the improvement versions of BioBrick Part: ahpC promoter (Part:K362001) designed by [http://2010.igem.org/Team:KIT-Kyoto/Parts 2010 KIT-Tokyo team]. AhpCp2D1 (Part:BBa_K1104203) was used as sensor to ROS (Reactive Oxygen Species). More details about ROS-induced promoters and part improvements of ahpC promoter are in page:[http://2013.igem.org/Team:NYMU-Taipei/Project/Inhibition/Sensor Sensing Nosema ceranae].

AhpCp2D1 +E0840 (Part:BBa_K1104244)

The reporter: AhpCp2D1 +E0840 (Part:BBa_K1104244) includes promoter (Part:BBa_K1104204) and Part:BBa_E0840. We induce the promoter by different concentrations of H2O2 to identify the strength. After AhpCp2D1 (Part:BBa_K1104204) is activated, GFP generator BBa_E0840 takes as input a transcriptional signal (PoPS) and produce as output the fluorescent protein GFP.

We tested the improved promoter: AhpCp2D1 by following these [http://2013.igem.org/wiki/index.php?title=Team:NYMU-Taipei/Experiments/Protocols&oldid=243384 Promoter Testing protocols]. This wiki page shows the protocols we used for promoter testing.

Promoter Testing

These are the protocols we used for promoter testing

General Gene Reporter Assay protocol

This is our general setup for gene reporting assays.

Previous-night Preparation (~10m-60min)

Previous night:

  • Culture overnight (~16hrs) at 37C and shaking at 180rpm 2-3ml of single colonies of things to be measured.

Preparation (can be done the previous night too):

  • Determine the number of measurement points = number of items x number of replicates
  • Prepare that amount of 15ml round-bottom tubes and 1.5uL microcentrifuge tubes.
  • Add 2ml of LB + the relevant antibiotics in each round-bottom tube.

Pre-start (~30mins)

Preparation:

  • Prewarm the LB+antibioics to the temperature to test at (e.g. 20mins prewarming in the incubator takes it up to 37C).
  • Get some ice buckets and pre-cool down the 1xPBS

Culturing (2~3mins)

  1. Add any relevant chemicals to the prewarmed round-bottom tubes containing 2ml LB.
  2. Dilute the overnight culture to an OD600 of about 0.0325
  3. Incubate at the required conditions (usually 37C and 180rpm).
  4. Repeat for every replicate.

Measurement point retrieval

For each culture tube:

  1. Take out the relevant culture and put it on ice
  2. Extract 1.3ml from the culture tube and put it in an 1.5uL microcentrifuge tube
  3. Centrifuge for 1min at 16.2g (13.2rpm on tabletop centrifuge)
  4. WASH: Tip out the supernatant and add 650uL of 1xPBS, resuspend, then centrifuge again.
  5. WASH again.
  6. Carefully remove and discard all the supernatant, resuspend in 1.3ml of 1xPBS.
  7. Store at 4C until measuring

Measuring

Requires black plates for measuring fluorescence and transparent plates for measuring OD600. For each measurement point, load 200uL each into 3 black wells and 3 transparent wells (i.e. 3 technical replicates each for fluorescence and OD600). Measure the fluorescence on the black plate with the settings depending on your machine. We use excitation/emission wavelength of 485/525 (100ms per measurement). Measure the OD600 on the transparent plate.

H202 Promoter Gene Reporter Assay

For measuring promoters under different H202 stress concentrations: (e.g.: 0.01mM, 0.05mM, 0.1mM, 0.5mM, 1mM, 2mM, 2.5mM, 5mM).

  • Add the H202 just before adding the bacteria.

For reporting assays using 2ml round-bottom tubes:

  • First serially dilute the relevant concentrations from the stock (35%) everytime the experiments start.
  • Add 13.4uL of the relevant concentration of H202 into the tube.
35% 3.5% 1.75% 0.875% 0.7% 0.35% 0.175% 0.035% 0.0035% 0.00175%
5mM 13.4
2.5mM 13.4
2mM 13.4
1mM 13.4
0.5mM 13.4
0.1mM 13.4
0.05mM 13.4
0.01mM 13.4

Results

Relative promoter strength testing

This is the relative promoter strength of BBa_K1104204 under different H202 concentrations tested using BBa_K1104244 (BBa_K1104204 +E0840) and found that there is a trend where the higher the H202 concentration, the higher the promoter expression:

NYMU AhpCp2D1 strength.png

Strengths of the basal level of various promoters that respond to reactive oxygen species with respect to BBa_J23101 :

Comparison between ahpC and its improvements. (Part:BBa_K1104244)

Promoter testing of ahpC promoter (Part:K362001) and its derived promoters.

NYMU AA.png
NYMU AAA.png
NYMU AAAA.png

Device1 testing

OxyR-included circuit(Device1+Device2)

In OxyR-included circuit, Device2 is composed of sensor (OxyR-induced promoter, including TrxCp, HemHp, sufA, AhpCp1000, AhpCp2D1, AhpCp2, AhpCpD1, AhpCp1, DsbGp) plus reporter (Part:BBa_E0840). In this case we use AhpCp2D1.

Device1 (Part:BBa_K1104250)
a constitutive promoter(Part:BBa_J23102)+ RBS(Part:BBa_B0034)+ activator OxyR(Part:BBa_K1104200)+ double terminator Part:BBa_B0015
Device2
a sensor (OxyR-induced promoter, including TrxCp, HemHp, sufA, AhpCp1000, AhpCp2D1, AhpCp2, AhpCpD1, AhpCp1, DsbGp) + reporter (Part:BBa_E0840)

Device1 in order to enhance the effect of ROS on E. coli is added ahead: a constitutive promoter(Part:BBa_J23102), RBS(Part:BBa_B0034), activator OxyR(Part:BBa_K1104200),and double terminator Part:BBa_B0015.

Putting Device1 device:BBa_K1104250 upstream to promoter AhpCp2D1(Part:BBa_K1104204) remarkably enhanced the strength of promoter AhpCp2D1(Part:BBa_K1104204) as our experiment results shows:

NYMU 2D1O.png
NYMU 2D1Ovs.png