pT7_RBS_xyIE reporter gene : BBa_K784001
A big part of our project was the to create a colorimetric signal. To achieve that we expressed the gene XylE, into the enzyme Catechol 2,3-dioxygenase (CDO), using the cell free system OnePot PURE. When CDO reacts with the substrate catechol transforms into another product, which causes a color change from transparent to yellow.
Since we did not have this specific part on our stock, we used part
A color change is observed in the reaction; after 30 minutes of incubation the color starts to become yellow and after 1 hour the color is bright and distinguishes clearly from the controls. Both controls remain transparent throughout the runtime. So we can conclude that the gene XylE with a T7 promoter was successfully expressed in the cell free system OnePot PURE into its corresponding enzyme, CDO. Initiation factor 1 : BBa_K2443063 IF1 is one of the three initiation factors that are needed for translation in bacteria. In addition it is one of 36 proteins that produce the PURE cell-free system. We expressed this protein using a T5 lac operon instead of the T7 that was used by team iGEM17_Lethbridge to achieve better regulation. IF1 has a molecular weight of around 9kDa and it is the only one of the PURE proteins with such low weight. Because of that it is easy to distinguish its corresponding band on a SDS-PAGE gel. The following image was produced according to our protocol(link) and shows the results of SDS-PAGE for the OnePot PURE protein solution:
It is clear that we successfully expressed IF1 using a T5 lac operon promoter.
During our project we introduced many parts to the iGEM community, mainly relevant to the OnePot PURE cell-free system. In this section will present two of them : Thermal unstable Elongation Factor (EF-Tu), both the coding sequence equipped with a HIS affinity tag-BBa_K2916009 and the expression system in E.coli -BBa_K2916038 and the expression system in E.coli of Nucleotide diphosphate kinase (NDK) - BBa_K2916047 EF-Tu is an indispensable component in the PURE cell-free translation-transcription system, catalyzing the binding of an aminoacyl-tRNA (aa-tRNA) to the ribosomes. It plays a crucial role in the OnePot PURE cell-free system as it accounts for 47% of the total protein concentration in the protein solution. The sequence includes a 6x HIS affinity tag, to allow the purification of the expressed protein while the composite part optimized for expression in E.coli bacteria has a T5 Lac operator, which allows us to regulate the expression of this protein. On the other hand NDK plays a big part in the energy-recycling system. In particular NDK catalyzes the reactions of phosphorylation of a histidine residue that produces NTP, which then initiates a chain of reactions in the system. Analyzing those reactions we can conclude that NDK is involved in the synthesis of nucleoside triphosphates (NTP), such as guanosine triphosphate (GTP), cytidine triphosphate (CTP) and uridine triphosphate (UTP) that are all parts of our energy solution. The characterization of the parts was made in two steps. First we tested if the protein was actually expressed and purified as intended. We measured the molecular weight of EF-Tu at 43kDa and 17kDa for the NDK and we can clearly see their corresponding bands on the gel presented in the previous section The next step was to prove if they are actually functional. The experiment was an indirect one. For the OnePot PURE system to translate and transcribe efficiently it is necessary for all 36 different proteins to be present and fnctional, meaning that a successful outcome would verify the fine performance of EF-Tu and NDK, while an unsuccessful one would lead to inconclusive results. On the graph bellow you can see the expression of sf GFP in OnePot PURE and PURExpress with DNA template of 5nM:
Since the protein is expressed we can conclude that the two proteins, along with the rest of the components, function as desired.
Toehold switch for Bois Noir 2.1 with superfolding GFP: BBa_K2916073 During the development of our project, we decided to improve the "superfolder GFP driven by T7 promoter" : BBa_I746909 As we wanted to produce a signal only in case of the presence of the wanted sequence, we needed to find a way to regulate the protein expression in our system. We achieved that by adding a Toehold, that has an integrated T7 promoter and a Ribosome Binding Site, before the encoded gene sequence. This procedure blocks the expression of sf GFP in absence of trigger DNA and and allows its expression only when the specific sequence is present in our sample. With our improvement, sf GFP can now be used as a diagnostics tool for detection of Bois Noir disease in the grapevine. This part was created with PCR. Specifically, we acquired the existing part in the distribution kit, and removed the promoter and RBS sequence. Then we run PCR with primers that had the sequence of the toehold already build-in. We tested the functionality of the new part by expressing it in OnePot PURE cell-free system (with and without triggering DNA) and measuring the excitation at wavelength of 535nm in a platereader.
There is some leakage present in the final results,but given that the output of the reading when the wanted sequence is present is more than 5-fold larger there can be a clear distinction between the ON and OFF phase of the toehold