Difference between revisions of "Team:KU Leuven/Wetlab"

We designed a circuit capable of forming patterns in a controlled way. Using a modified and temperature-sensitive lambda repressor (cI), we can trigger formation at desired points in time. This time-dependent controllability, together with the possibility to change many different parameters and output signals, leads to an enormous tunability in the creation of the patterns. Our mechanism will stimulate advancements in a variety of industrial processes like the creation of novel bio-materials. This fundamental project could also speed up medical research projects like tumor formation and tissue regeneration.

Basic components

Two different cell types, called A and B will interact and create patterns. In order achieve the desired behavior, the cells used in our experiments will not be wildtype Escherichia Coli, but specific knockout strains. Celltype A has a deletion of tar and tsr, whereas in celltype B, both tar and cheZ are knocked out. Tar and Tsr are two major chemotactic receptors and cheZ is a key phosphatase that regulates cell-motility. In absence of both Tsr and Tar, the cell is rendered insenstive to leucine as a repellent and attractant. On the other hand, cells who have lost only Tar, are repelled by leucine. In cell B, the absence of cheZ causes incessant tumbling.

Both cells are transfected with only one plasmid (figure 1). Each plasmid contains a temperature sensitive cI repressor protein, which is constitutively expressed. This repressor is only able to bind to the lambda promotor at temperatures below a certain threshold. At elevated temperatures, cI is unable to bind to the promotor region enabeling RNA polymerase to start transcription. From the lambda promotor in cell A, two essential protagonists for our system are expressed: The AHL- producing enzyme LuxI and the transcription activator LuxR. On the contrary, cell B only contains the lambda promotor coupled to the LuxR gene. Cell A fulfills a dual role by coupling the expression of an auto-aggregation factor Ag43-YFP and a leucine-producing enzyme IlvE to the AHL-LuxR regulated promotor. Ag43 causes cells A to stick together and form clumps. At the same time, IlvE produces the repellent leucine. The repellent has no impact on cell A, since it does not contain the necessairy receptors anymore. Cell B on the other hand is repelled by leucine, and contains a Lux promotor coupled to CheZ-GFP and the transcription repressor PenI. This repressor binds to the PenI-promotor, which regulates the transcription of a RFP. In order to measure the concentration of the key proteins LuxI and LuxR, they were fused with a His-tag and an E-tag respectively. To ensure rapid degradation of proteins whose expression is AHL concentration dependent facilitating the readout, an LVA tag was fused to CheZ, GFP and RFP.

Lexicon

Temperature sensitive $$cI_857$$ : Mutated variant of the phage lambda repressor protein. It can bind firmly to the lambda promotor and represses transcription at low temperatures, but is unstable at elevated temperatures.
LuxI = enzyme for production an AHL called oxohexanoyl-homoserine lactone. This highly diffusible molecule is used as a communication molecule between cells, and is able to bind with LuxR at the lux promotor to initiate transcription.
LuxR = cytoplasmic receptor for AHL
Ag43 = membrane protein, causes autoaggregation of cells
IlvE = last enzyme in the biosynthesis of leucine. In order for it to work, we need to provide the right substrate:4-methyl-2-oxopentanoate to the medium.
Leucine = amino acid, but also a repellent if tar is knocked out
GFP = green fluorescent protein
YFP = yellow fluorescent protein
RFP = red fluorescent protein
PenI = Repressor protein, binds to the PenI regulated promotor
LVA = degradation tag, signals the linked protein for intracellular degradation
His-tag: repeat of six histidines, in order to purify it with nickelbeads
E-tag: links the tagged protein with an anti-E antibody