Team:Slovenia/METHODS and PARTS/parts

Fun fact:

We have deposited 151 new parts to Registry, 135 of them being in pSB1C3 vector.

Our parts can be divided into several groups:

• DNA binding domains: We deposited five new zinc fingers and DNA-binding protein TAL. Most of them are well characterised and supported by different experimental techniques. They are also prepared with an extension linker making them useful for any further modifications. Some variants also contain His tags for quick isolation and purification.
• Split GFPs with overlapping amino acid extensions at the reassembly point are a useful tool for dimerisation and binding studies.
• Chimeric biosynthetic enzymes: A principle of binding biosynthetic enzymes to our submitted DNA program with fused zinc finger domains have a potential for production of various different metabolites and increased speed/yield of reaction.
• In vivo testing devices: Parts composing a device for testing in vivo binding of DNA binding proteins to their target sequences. 
• Oscillator: Parts for composing oscillators that function both in pro- and eucaryotic cell systems. 
• DNA program: We deposited several DNA programs used in DNA binding studies as well as in biosynthesis pathway experiments.

We selected a few most important and therefore favorite parts:

• Biosynthetic device

We fused violacein producing enzymes to zinc fingers and successfully proven that they retain their function (BBa_323132 and BBa_323135). Furthermore after adding a DNA program (BBa_323066) we demonstrate a 6-fold increase in violacein production and elimination of unwanted side products (see our Results).

• Device for testing binding of DNA binding proteins to their target sequences in vivo

To test whether binding of zinc fingers or TAL to a corresponding specific target DNA sequence occurs in vivo, we designed a device composed of several parts:

1. a synthetic promoter pSYN in which a DNA binding sequence, particular for each zinc finger to be tested, was inserted between -35 and -10 sites using BbsI restriction site (DTER_pSYN_BbsI, part BBa_K323088)
2. lacZ reporter gene, which expression is controlled by pSYN
3. DNA binding protein (cut with XbaI/NotI) to be tested under arabinose inducible (pBAD) promoter in lacZ_DTER_pBAD_BsaI_DTER cut with BsaI (BBa_K323089)

 The successful binding of DNA binding protein to the synthetic promoter would prevent transcription of lacZ resulting in lower beta-galactosidase activity. 

 

• Split GFP/FRET device



Combining five BioBricks, four of them being proteins, and one being an non-codable DNA program sequence (BBa_323039), mammalian cells exhibit split mCerulean and split mCitrine reconstitution upon binding predefined DNA elements. When both subsystems bind DNA, the composite device is ready to FRET. Original BioBricks had to be cloned into pSB1C3 backbone thereby loosing mammalian terminator. We have deposited both sets of four functional plasmids, one set being cloned into desired submission backbone (parts BBa_K323021, BBa_K323029, BBa_323005, BBa_323080) and the other set of plasmids originating from pSB1AK8. 

 

We characterized several parts already deposited in Registry:

 

• pBAD/araC promoter (Part:Bba_I0500)

We further characterized the pBAD/araC promoter (Part:Bba_I0500) using lacZ gene that encodes for beta-galactosidase enzyme (Part:Bba_K323133). When incubating E.coli cultures containing the lacZ under pBAD/araC promoter, increasing concentrations of arabinose leads to higer promoter activity that results in higher amounts and subsequently higer activity of beta-galactosidase enzyme. The tested cultures incubated at different concentrations of arabinose (i.e. 0%, 0.001%, 0.0025%, 0.005%, 0.01%, 0.025%, 0.05%, 0.1%, 0.25%, 0.5%, 0.75% and 1%) have shown a trend of increasing beta-galactosidase activity with raising arabinose concentration. The activity was highest at 1% added arabinose, which implies the promotor should be induced by this arabinose concentration in order to reach maximum expression of the regulated protein. However, some beta-galactosidase activity was observed even at 0% added arabinose, which indicates slight leakage of the pBAD promoter.

 

• Zinc finger protein Gli-1 DNA-binding domain (BBa_K165007) from Brown University in iGEM 2008.

We added His tag sequence at N-terminal side and N-terminal split CFP sequence at C-terminal side of Gli-1 DNA-binding domain and deposited it as Gli1_link_nCFP (BBa_K323016), cloned it under T7 promoter for production in E. coli BL21(De3)pLsS strain and deposited it as BBa_K323058. We enclosed a detailed description of Gli-1 back to its original datasheet, results of its isolation and purification (SDS and Western blot, CD analyses) and characterize its binding properties with several binding assays (binding in vitro to its target DNA sequence monitored with SPR, binding in vivo tested with beta-galactosidase assay).

 

• Zif268-HIV DNA-binding domain (BBa_K165006) from Brown University in iGEM 2008.

We added His tag sequence at C-terminal side and C-terminal split CFP sequence at N-terminal side of Zif268-HIV DNA-binding domain and deposited it as cCFP_link_HivC (BBa_K323071), cloned it under T7 promoter for production in E. coli BL21(De3)pLsS strain and deposited it as BBa_K323069. We renamed this part into zinc finger HivC (a mutation variant of Zif268 zinc finger) according to work done by Reynolds and co. (2003). We enclosed a detailed description of HivC back to its original datasheet, results of its isolation and purification (SDS and Western blot, CD analyses) and characterize its binding properties with several binding assays (binding in vitro to its target DNA sequence monitored with SPR and EMSA assay, binding in vivo tested with beta-galactosidase assay).

 

• Vio operon (BBa_K274002) from Cambridge in iGEM2009

We analysed the products of violacein operon by thinlayer liquid chromatography and mass spectrometry. Results of our experiments show that violacein operon produces functional enzymes that synthesize three main compounds, namely violacein, deoxyviolacein and deoxychromoviridans (this was confirmed by comparing compounds with violacein standard and mass spectrometry). BBa_K274002 produces a large amount of deoxyviolacein and smaller amounts of violacein and deoxychromoviridans. Experimental data were deposited back to BBa_K274002 datasheet.

 

 

Our biosynthetic device demanded transformation of three deferent plasmids to E. coli cells. This is only possible, if these plasmids carry different antibiotic resistence. So cloning and biobrick assembly required careful planning of cloning strategy. Altogether we made more than four hundred parts. For the purpose of Registry deposition we choose most important 135 parts and inserted them in pSB1C3 backbone.

 

Check our sandbox and favorite parts. 

 

Reference:

Reynolds L., Ullman C., Moore M., Isalan M., West M.J, Clapham P., Klug A., Choo Y. 2003. Repression of the HIV-1 5 LTR promoter and inhibition of HIV-1 replication by using engineered zinc-finger transcription factors. PNAS (2003)