Regulatory

Part:BBa_K1104204:Experience

Designed by: Ting-Yun Chiang   Group: iGEM13_NYMU-Taipei   (2013-09-16)

Introduction

This part: AhpCp2D1 is one of the improvement versions of BioBrick Part: ahpC promoter (Part:K362001) designed by 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: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 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:

Error bars represent the 95% confidence interval

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

Comparison between ahpC and its derived promoters by setting J23101 as 1(Error bars represent the 95% confidence interval) (Part:BBa_K1104244)

Promoter testing of ahpC promoter (Part:K362001 ) and its derived promoters.The figure suggests that AhpCpd1 and AhpCp1 show so weak promoter strength that we only focus on the change of promoter strength of the three promoters as following:

The promoter strengths are standardized by setting AhpCp1000 in the absence of H202 as 1
The strength of the three promoters are compared to their respective promoter under the H202 concentration 0mM and 1mM

Considering both the promoter strength and the effect of H202, AhpCp2d1 is later chosen as the OxyR-induced promter for our testing.

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:

(Error bars represent the 95% confidence interval)
In the presence of Device 1, AhpCp2D1 shows 7 to 9 folds of strength (Error bars represent the 95% confidence interval)

Future planning

From the above figure which shows relative promoter strength of AhpCp derived promoters, we can till the similar riding trend of promoter strength in response to H2O2 concentration. We assume that it's due to the fact that the promoters have same ribosome binding site; as a result, in order to future improve and regulate the promoter strength, we can try to focus on enhancing the strength of ribosome binding site or increasing its number.