Team:KULeuven/Details

Extended project description

still under construction: Clarifying pictures will be added soon, all biobricks will be clickable and lead towards the biobrick register

This summer, we are engineering the bacterium E.D. Frosti that induces ice crystallization, using the ice-nucleating protein ( INP), or will inhibit ice crystal formation, using the anti-freeze protein ( AFP), depending on the given stimulus. These proteins will be extracellularly anchored at E.D. Frosti’s cell membrane. Furthermore, it is essential to create a dual-inhibition system, so that INP and AFP can never be expressed at the same time. To verify the function of this system, we coupled the production of a specific color depending on the stimulus given to E.D. Frosti. Finally, as a safety mechanism, we installed a suicide mechanism in E.D. Frosti, whose activity is mediated by an “AND”-gate system: the cell death mechanism is only activated when one of the two stimuli is given, AND a sudden decrease in temperature (a cold-shock) occurs.
To realize our E.D. Frosti project, we had to create several mechanisms which will be outlined below.

1. Dual inhibition system

Since E.D. Frosti has all the genetic information to exercise both functions, it is very important to make sure that only one type of proteins will be expressed in one E.D. Frosti cell. A dual inhibition system is a good way to ensure this and is explained in the next figure.
When arabinose is added to the medium (stimulus 1), it will induce transcription from the pBAD promoter ( BBa_I13453 ), resulting in LuxR ( BBa_I0462 ) and LuxI ( BBa_C0261 ) transcripts that are translated into the respective proteins. LuxI is an enzyme that catalyzes the production of N-Acyl homoserine lactones ( AHLs ) from S-adenosyl methionine (SAM ) and acyl-coenzyme A (acyl-CoA). These AHLs, when bound to LuxR, are able to regulate transcription through binding with a luxR binding site located in promoter regions. In our system, the LuxR-AHL complex will perform a dual task.
While it activates the pLux-CI promoter ( BBa_R0065), resulting in the transcriptional activation of OmpA-AFP (NEW BIOBRICK)and MelA ( BBa_K193602 ) (2), it acts as a negative regulator of the pLac-Lux promoter ( BBa_K091100), the transcriptional regulator of stimulus 2, an additional safety mechanism to ensure that no INP can be produced during stimulus 1, even under non-inducing conditions. If we add lactose or IPTG to the medium (stimulus 2), it will activate transcription from the pLac-Lux promoter , resulting in INP (NEW BIOBRICK), CI repressor and CrtEBI ( BBa_K274100 ) transcripts that are translated into their specific proteins. The CI repressor will repress the pLux-CI promoter and, thereby specifically inhibiting the production of AFP, while INP is being expressed.