Team:UNIK Copenhagen/Green Lab
Green Lab
What is Green Lab about?
For the competition a required output of our synthetic biology work in the laboratory is the creation or improvement of a so called “BioBrick”. A BioBrick is a Lego-like building block that is used to design and assemble synthetic biological circuits. This is a gene created from scratch that can be inserted in a number of different biological organisms such as E.coli, moss and tobacco in order to produce components or perform functions that the organisms would normally not do: such as glow yellow and produce an antioxidant such as resveratrol. For our project we will both improve and create a BioBrick which will be sent to the iGEM headquarters and become part of their “BioBrick registry”. This is basically an open source library of genes that can help speed up the development of synthetic biology by making genes accessible to all. We will create a new BioBrick containing an antifreeze protein gene and improve on a BioBrick containing the resveratrol gene.
Outside the scope of the competition we will insert the DNA gene containing YFP and an antifreeze protein gene and a resveratrol gene. When this is transformed into the moss we should have an output of moss that is able to produce the antifreeze protein on its own, or produce resveratrol. The moss will also glow yellow due to the YFP when it is producing either of the components, which acts as a proof of concept.
Technical description
We will make a stable and transient transformation of moss, Psychomitrella patens. We will make two linear gene constructs for stable transformation in moss. One construct is going to contain a gene encoding an antifreeze protein and the other construct will contain a stilbene synthase-gene (STS), making the final enzyme in the resveratrol pathway. Both constructs contain regions homologous to the moss genome, so they will integrate via homologous recombination in moss. The constructs furthermore contains the ZmUbi-promoter, resistance gene and YFP, to confirm transformation. We will also make a transient moss transformation with a vector containing the ZmUbi-promoter followed by YFP, to confirm the function of the promoter in moss.
Due to time constraints, we will also transform tobacco and E.coli. We will transform tobacco with the STS-gene, using USER-cloning and Agrobacterium-mediated transient expression, in order to quickly confirm the functionality of the gene (moss takes a long time to grow!) by measuring the presence of resveratrol with Liquid chromatography-mass spectrometry. Furthermore we will transform E. coli with the antifreeze gene which originate from the spruce budworm, to quickly see whether the gene has an effect on temperature sensitivity. We will perform experiments at low temperatures to measure the growth of the bacteria when exposed to cold. We will also make a his-tag version of the antrifreezeprotein to confirm the presence of the protein by western blotting.
One of our petri dishes with moss
Moss growing facility