Team:UAM Poznan/Practices

HUMAN PRACTICES

Biology and molecular biology classes

Our constructs, originally containing his-tag, will be also designed with other tags and used for educational purposes, for biotechnology and molecular biology students during their laboratory classes. Our promoters will be useful for the controllable turn on of E.coli genes expression, and also for testing and designing systems for better purification of recombinant proteins produced in E.coli.

Poznań Festival of Science and Art

Our team members took part in a series of workshops prepared for high and middle schoolers. We showed them how to isolate DNA from kiwi, taught them about DNA and cells, also organized a biology related quiz! However, the main attraction was a show with a liquid nitrogen. You can find the photos from this event on our event page.

7^th Conference of Polish Society of Experimental Plant Biology

Our group showed the results of experiments on promoters induced by sugars on the 7th Conference of Polish Society of Experimental Plant Biology and the Intercollegiate Faculty of Biotechnology UG & MUG, on the "System and Synthetic Biology" session. The conference took place in Gdańsk, 8-11 September 2015. We prepared two posters - one about the CPEC method and one about the assembly and activity check of our promoters - and also an oral presentation, which was given by Marcin Osuch. The presentation was awarded with a diploma for the best student oral presentation. The conference gave us the opportunity to introduce ourselves to the great scientist from all over the world and receive many kind suggestions for our later work. You can find the photos from this event on our event page.

Below the abstracts of our posters from the event:

EcoFactory, a multipromoter explorer expression system for Escherichia coli

Minimal, independently regulated promoters of different expression levels are important tools for synthetic biology to build complex but compact genetic systems. It is important for crystallographers to produce recombinant proteins of high purity and for the pharmaceutical industry to make the whole process free of toxic compounds. Ideally, the expression level should be tuneable, induction - non-toxic, mRNA translation – free of mistakes, with perfect folding; recombinant protein - soluble and homogenous, and finally its purification - fast and reproducible. We prepared a series of plasmids expressing his-tagged superfolder GFP (Pedelacq J-D. 2006) under the control of four nontoxic inducers: arabinose, melibiose, rhamnose and xylose. The four promoters are more controllable than the most popular lactose induced T7RNA polymerase dependent systems. The coding sequences are transcribed by the cellular RNA polymerase which is slower and more accurate than T7 RNA polymerase. SfGFP can be used to compare activities of standard, modified, and minimal promoters during growth on different carbon sources and as a probe of a fused protein solubility. We present a comprehensive comparison of the sfGFP expression driven by inducible promoters with the insulated, constitutive promoters proC and proD as standards (Davis 2009). Two pairs of primers should be sufficient to clone an ORF of interest as a protein fused to sfGFP or to replace it in all vectors of the multipromoter system.

Authors: Zielińska J., Mokrzycka D., Lejman A., Szymańska M., Osuch M., Olejniczak O., Kopa M., Rabiasz A., Abramowski S. Bartoszewicz J., Nowicka M., Rżosińska K. and Nuc P.

Towards the simple and uncompromised assembly of genetic systems

An underlying necessity of synthetic biology is the ease, simplicity and possible automation of assembly of genetic systems. In 2009 J. Quan and J. Tian have developed the method for an efficient construction of combinatorial libraries (CPEC: circular polymerase extension cloning). At the same time D. Gibson and his co-workers from Craig Venter Institute have described another restriction site independent method of cloning and de novo assembly of large genomes from overlapping DNA fragments with a mixture of three enzymes: T5 exonuclease, Phusion DNA polymerase and Taq DNA ligase. The cloning system as well as the nebuilder software for design of the overlaps are available from New England Biolabs. Although Gibson Assembly is the method of choice in construction of larger genetic assemblies, we have found that CPEC can be successfully used for the assembly of smaller genetic systems composed of up to 5 DNA fragments with overlaps constructed with the nebuilder software. This cloning system is more flexible and faster than building more complex constructs from biobricks (2011 Shetty), more precise, more efficient, and cheaper than Gibson Assemby. Construct assembly can be completed in 2 -3 days by two rounds of PCR and transformation of electrocompetent cells. From a standard cloning we usually obtain several hundreds of colonies of which almost all contain a construct of the expected assembly and sequence. The expression system is under development for fast 15-min. purification of soluble recombinant his-tagged proteins in Escherichia coli.

Authors: Zielińska J., Mokrzycka D., Lejman A., Szymańska M., Osuch M., Olejniczak O., Rabiasz A., Kopa M., Abramowski S. and Nuc P.

Night of the Scientists

Constitutive promoters assembled for igem competition will be used during our lab shows on the Night of Scientists, that will take place on the 25^th September 2015 at our University. Participants will work with sfGFP under proC promoter and perform protein purification. Our team members will explain processes connected with producing recombinant proteins in E.coli.