Team:UAM Poznan/Demonstrate
APPLICATIONS
We are developing the multipromoter explorer expression system for Escherichia coli for several reasons, and we believe that it will be useful, and to some extent it already is.
1. This is quite an usual problem in molecular biologists’ life that she or he is trying to produce a functional protein quickly and cheaply and the first choice is usually to start from an available commercial expression system for E. coli. Unfortunately in many cases this simple task starts to be problematic and terribly time consuming. We want to help! a) the most popular T7 polymerase-based pET or pET-like expression systems are leaky. After experiments and tests we did, we know why those systems are leaky: once produced T7 polymerase is quite active and stable. This is known as well, that even very tightly repressed promoters leak, at least because genes located in genome from time to time undergo replication and repressors have to be released for a while. Then production of even slightly toxic proteins starts to be very difficult. In the case of a recombinant protein production failure, molecular biologist is trying to check if the induction of expression is working, if the protein is soluble, if the reading frame is O.K. if the host strain is the best choice and so on. Waste of time! It is much better to have positive controls we provide, i.e. easily detectable, soluble fluorescent protein under promoters induced by rhamnose, arabinose, melibiose and xylose. Cell lysis buffer (it’s composition) is the next suspected for our failure in recombinant protein production. Having a positive control with a fluorescent or chromogenic protein (under construction) one can easily check how efficiently cells are disrupted and what is the efficiency of recombinant protein recovery on affinity chromatography column of one’s choice. Our control constructs produce superfolder-GFP with a 6-Histidine tag at the N end. So all of them can be used to check buffers and chromatography columns before one decides to run purification of her/his precious, usually more difficult protein.
2. All above mentioned traits of this system make it useful for laboratory classes in molecular biology and several of them are already in use during practicals at our University. Our constructs were also used during our workshops organized for high school students (see our Human Practices page)
We further develop the system by adding new chromatography tags, endopeptidase cleavage sites and export tags. We hope all of them will be useful educational tools for biology students at least at our University. We are grateful to iGEM community for sharing so many interesting constructs, we will be able to use in practical education.
3. The biggest achievement of our work, especially this summer, is the construction of a syntetic rhamnose induced promoter with E1 5’UTR (BBa_K2014001). We hope it can dominate protein expression in E. coli because:
a) Induced expression is higher than in a standard T7 based system in which over 90% of protein expressions worldwide are performed.
b) Is much more tightly controlled than T7 systems, so it should be easier to produce proteins harmful for components of E. coli cells.
c) E.coli RNA polymerase which drives this system is more accurate and slower than T7 RNA polymerase of bacteriophage origin. Accuracy is important for recombinant protein production for medicine and for crystallography. Slower polymerase – means less problems with mRNA secondary structures.
The system works perfectly in real-world conditions.
Examples of GFP and RFP production in one of our expression vectors, with our pBAD M 5’UTR promoter (and this is not the strongest one! BBa_K2014003).
The protein extraction and purification procedure can be tested with a positive control of one’s choice:
GFP and RFP are not denatured during SDS/PAGE electrophoresis so both can be visualized in gel documentation systems under UV light:
Every construct we provide has been fully sequenced and thoroughly tested.
