Team:Imperial College/Constitutive

Aims

• To characterise the constitutive promoter and RBS combinations. This protocol is concerned with characterisation of the steady-state expression of GFPmut3b under the control of the 4 different promoter-RBS combinations. We will be measuring the fluorescence and O.D.₆₀₀ of the cultures and normalizing our data to the units of rate of molecules of GFPmut3b synthesised per cell per min/sec.
• In addition, from comparison of the rate of expression we should be able to determine the relative strength of the RBS.
• With the data on the characterisation of the promoter-RBS combinations, we will be able to make an informed decision on which combination we should use for our future test constructs.
• The protocol below describes what is needed for one combination of promoter and RBS, we will need to carry this out for all the combinations.
• Normally we are only concerned with the steady state expression of a constitutive promoter. However, the conditions under which we measure this could change, for example what media the B.subtilis is grown in. In addition, we may wish to see how the strength of expression changes during the growth of the B.subtilis. This could be achieved by measuring the change in fluorescence between the different growth phases.

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

Protocol

Day 1

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

Erika says:

Work out how many cultures you are going to need (nine by the looks of things below!) and do the above nine times...

J-We can start with just one overnight culture then make inoculate 3 x fresh media in the morning. The reason for 9 is that for each of the cultures we make on day 2, we pipette 3 aliquots from it. This helps us to average for any pipetting errors when the plate is loaded, whereas the different cultures help to give independent repeats for the experiments.

Day 2

1. Collect 3 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 LB blank). Remove 1ml of the overnight culture and measure the O.D.₆₀₀
2. Dilute the overnight culture 1 in 10 (2ml per culture) in 3 x 20ml 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 9x200μl of the B. subtilis into a 96 well plate following the plate schematic (in total want 3 repeats for each of the 3 cultures). In addition pipette 200μl of 3xLB media into the 96 well plate. Once the plate has been loaded then carefully place sticky tape onto the top of the plate.
4. Place into the plate reader and open the protocol for characterisation of constitutive promoters and run protocol.
5. This needs to be set up to measure the O.D.₆₀₀ and fluorescence (488nm excitation filter and 525nm emission filter) every 10 minutes for an hour (What do we think of these data collection settings).
6. Once data is collected dispose of the 96 well plate into the autoclaved rubbish bins (white).

Erika says: Can we not inoculate and put straight into the plate reader? Why do we have to wait until exponential phase?

J-Yeah we can put it in straight away and see what happens. My only concern is that I do not know how happy the bacteria will grow in the plate. The population is small but the available media is limited. Clearly this needs to be determined.

Can the plate reader measure OD? That would be handy. -Yes we measure the O.D.₆₀₀