Team:Imperial College/Inducible

Aims

• The aim of this protocol is to help us define the dynamic range of the transfer function where the lower and upper threshold response is. This can help us define further concentrations to characterise the transfer function. In addition, we can get an idea of the response time of this inducible promoter. Finally if we need to pursue with cloning these experiments will confirm which of the combinations work and can be pursued for cloning further constructs.
• This protocol is to be used to characterise the IPTG inducible Hyper-spank promoter RBS combinations.

Equipment

• Fluorometer + PC
• Pipette gun
• Gilson p20,p200,p1000
• Spectrometer
• Shaking incubator

Reagents and Materials

• 1 x 96 well Fluorometer Plate (See-through bottom)
• Sticky plate lid
• Cuvettes
• 4x20ml of Autoclaved LB media in a 200ml flask Containing suitable antibiotics
• IPTG

Protocol

Day 1

1. Collect 10ml of LB media (containing suitable antibiotics) into a 100ml flask. Innoculate the media with a single colony from a B. subtilis plate and grow overnight at 37^o.
   

Day 2

1. Collect 1 x 200ml flasks containing 20ml of LB media (containing suitable antibiotics) and remove 1ml of the media and pipette into a blank cuvette (this is to make the blank for OD measurements). Remove 1ml of the overnight culture and measure the OD₆₀₀
2. Dilute the overnight culture 1 in 10 (2ml in 20 ml) in 1x20ml of fresh LB media containing suitable antibiotics . Mix thoroughly and remove 1ml of the culture and measure the OD₆₀₀. Place into a shaking incubator and grow until reaches the exponential phase of growth, the time for this should be determined previously when we do the growth curve but always checked by removing 1ml of each culture and measuring the OD₆₀₀ using LB media as a blank.
3. Once the correct OD₆₀₀ has been reached then pipette 27x190μl of the B. subtilis into a 96 well plate following the plate schematic. In addition pipette 200μl of LB media and 190ul of LB media into the 96 well plate.
4. Now load in 10ul of the inducer into the plate following the schematic (need to be determined) with the concentrations listed below. In addition add 10ul of 10mM IPTG to the LB media alone, this will help us determine whether the IPTG is contributing to the background fluorescence.
5. Once the plate has been loaded then carefully place sticky tape onto the top of the plate.
6. Place into the plate reader and open the protocol for characterisation of inducible promoters and run protocol.
7. This needs to be set up to measure the OD₆₀₀ and fluorescence (488nm excitation filter and 525nm emission filter) every 10 minutes for 6 hours (What do we think of these data collection settings).
8. Once data is collected dispose of the 96 well plate into the autoclaved rubbish bins (white).

Erika says: What is the most frequent settings we can have on the plate reader? How long can we leave it? We can always do more concentrations of IPTG if we need to I suppose.

Concentrations of IPTG:

• 0x3
• 1nMx3
• 10nMx3
• 100nMx3
• 1μMx3
• 10μMx3
• 100μMx3
• 1mMx3
• 10mMx3