Team:KULeuven/Details

Extended project description

This summer, we are engineering the bacterium E.D. Frosti that induces ice crystallization, using the ice-nucleating protein (INP), or inhibits 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 test this system, we coupled the production of a particular color to a specific 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 construct our E.D. Frosti project, we had to create several mechanisms which will be outlined below.

1. Dual inhibition system

Since E.D. Frosti contains all the genetic information to execute both functions, it is important to make sure that only one type of protein is expressed in one E.D. Frosti cell. We found that a dual inhibition system is a good way to ensure this, as you can see 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 their 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 ( BBa_K584020])and MelA (BBa_K193602) (2), it acts as a negative regulator of the pLac-Lux promoter (BBa_K091100), the transcriptional regulator of stimulus 2, thereby acting as an additional safety mechanism to ensure that no INP can be produced during stimulus 1. If we add lactose or IPTG to the medium (stimulus 2), it will activate transcription from the pLac-Lux promoter , resulting in INP (BBa_K584024), 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.

2.a Expression of AFP and Melanin formation

If you read how the dual inhibition works, it already will be rather clear how AFP and melanin are produced: when E.D. Frosti takes up the arabinose, it will interact with pBAD and eventually triggers the transcription of OmpA-AFP and MelA. MelA will induce melanin formation, which gives a black color to our cells. In this way we know that our pLux-CI promoter is activated and that both OmpA-AFP and MelA are being transcribed and translated. The MelA is an existing BioBrick that the Cambridge_2009 team added to the registry: BBa_K274001 . For more information about MelA you can click here .

AFP normally is expressed intracellularly to prevent freezing of the cytoplasm, but in our system, it was essential that AFP was extracellularly anchored at the cell membrane, thereby preventing ice formation outside the cell. Therefore, we merged the AFP gene to OmpA via a flexible linker, thereby ensuring the desired extracellular localization of AFP. If you like to know more about AFP in nature and how it functions, you can click here.

2.b Expression of INP and Lycopene formation

INP and lycopene are produced as a response to lactose or IPTG uptake by E.D. Frosti from the medium. Lactose/IPTG will interact with the pLac-Lux promoter, so that transcription of CI repressor, CrtEBI and INP can occur. As discussed above, the CI repressor protein will act as an inhibitor of the pLux-CI promoter. The CrtEBI operon encodes the three last enzymes of the cartenoid pathway, that are needed to produce lycopene. E. coli itself is able to produce farnesyl pyrophosphate, which is converted to geranyl geranyl-PP by CrtE, which is transformed to pytoene by CrtB, which finally is modified to a lycopene molecule by CrtI. Lycopene production results in a bright red color, so that we can clearly see that CrtEBI as well as INP are expressed. Many different INPs exist in different organisms and we used the InaZ form from the Pseudomonas syringae. INP already contains a transmembrane domain and its active site is located extracellularly to induce ice nucleation in the environment. Therefore, we do not have to add an extracellular anchor. In fact, in previous iGEM competitions, the Berkeley_wetlab_2009 team has created a biobrick in which they specifically use only the (extracellular) INP-repeats as a cell-surface displayer spacer part, to improve the display of passenger domains: BBa_K197026 . If you like to learn more about INP, feel free to click here!

3. Cell death mechanism

We want E.D. Frosti to be coated with one of the two proteins, but we also want E.D. Frosti to die when we release it into the environment. Therefore, we created a suicide mechanism that is regulated by an AND-gate system. First, E.D. Frosti needs to respond to one of the stimuli, so that the pLac-Lux or the pLux-CI promoter is activated resulting in transcriptional activation.
The second part of the cell death mechanism works via a ribokey-ribolock system. The ribokey is under the control of the HybB cold-sensitive promoter (BBa_J45503), which will only be switched on when E.D. Frosti senses a dramatic drop of the temperature. So when the ribokey isn’t transcribed, the ribolock mRNA will form a hairpin structure, so that the ribosomes cannot access double stranded mRNA, and there will be no translation of the CeaB gene.
Hence, to activate the cell death mechanism, E.D. Frosti needs to undergo a cold-shock treatment. E.D. Frosti will ‘sense’ the change in temperature and the ribokey will be transcribed. This ribokey will bind to the ribolock, followed by the opening of the double-stranded mRNA hairpin loop. Under these conditions, ribosomes can bind and translate the CeaB gene. CeaB is a DNase, which can be found in the Colicin E2 operon (BBa_K131009 ). CeaB cuts all DNA in E.D. Frosti in small pieces, which results in cell death. We chose to only insert the CeaB gene instead of the whole Colicin E2 operon, since this also contains the CelB gene and the CeiB gene. CelB will induce cell lysis and under these conditions, E.D. Frosti would undergo complete degradation and this is something we want to avoid. We want E.D. Frosti to die, but it needs to retain its form, so that the proteins that are expressed on its cell membrane can retain their activity, even when the DNAse is active. The CeiB gene is an immunity gene, that results in DNase inhibition for the organism that is transcribing the CeiB gene.
So in summary, our cell death mechanism functions via an AND-gate system, in which both a stimulus and a cold-shock are needed to relieve the transcriptional and translational block, resulting in the death of E.D. Frosti. If you want to know more about bacteriocins or colicins such as CeaB, please click on this link.