Module Integration - Autoinduction

Overview

The autoinduction process switches on Module 2 following the accumulation of a predetermined concentration of polypeptide. The term 'autoinduction' was chosen as the flipping of this switch is triggered by the chemical composition of the media in which our chassis is grown.

The 'on signal' orginates from inside the system: hence 'autoinduction'.

Module 2 Timeline

Rationale

Before embarking on the creation of an autoinduction switch, we reviewed a number of possible alternatives. A genetically encoded timer was initially considered however these present a considerable number of problems that were absent from an autoinduction switch.

We believe that the advantages and modularity of our autoinduction switch is such that future iGEM teams will be able to deploy it in a broad range of senarios.

Initiation

Module 2 is triggered by a rise in levels of cyclic Adenosine Mono Phosphate (cAMP). This is because cAMP facilitates the dimerisation of the transcription factor CRP which induces transcription downstream of the PcstA promoter ([http://partsregistry.org/Part:BBa_K118011 BBa_K118011]). The rise in cAMP is correlated with a fall in levels of the primary carbon source (glucose). Thus, by controlling the initial amount of glucose present in the system, we achieve a timer function for the start of Module 2.

Module 2: Initiation

Results

Wet Lab

Module 2: Autoinduction Testing Construct

To characterise the properties of our autoinduction switch, we put the fluorescent protein GFP under the control of the PcstA promoter () as shown above.

Cells with BBa_K200018 were grown overnight in various different media, and the GFP fluorescence was measured.

After overnight culture, the corrected fluorescence of glucose is almost negligible, showing that glucose represses the PcstA promoter strongly.

For all the other secondary carbon sources, 10% Casamino Acids in M9 shows the highest corrected fluorescence at 22000.

  About the wetlab results!

Dry Lab

The first of our diauxic growth models is a cybernetic model developed by Kompala et al [1]. This model shows that glucose (S1) is used up before the secondary carbon source (S2). During this phase, the population (X) is in exponential grow until glucose (S1) runs out. This is followed by a stationary growth phase, and finally the population enters a second exponential growth phase as they start uptaking the secondary carbon source (S2). Another model was developed by Keasling [2] that takes into account the dynamics of the lactose operon.

  About the models and simulations!

Conclusion

We have chosen a strategy of autoinduction to control the initiation of Module 2. Based on experimental data collected by our team, we were able to select 10% casamino acids as the most suitable secondary carbon source for maximal output from the PcstA promoter.