Team:Slovenia/METHODS and PARTS/notebook/carot

6/7/2010 –6/13/2010

The following parts from registry were transformed into E. coli DH5α: [http://partsregistry.org/wiki/index.php/Part:BBa_K152005 BBa_K152005], [http://partsregistry.org/wiki/index.php/Part:BBa_K118000 BBa_K118000],[http://partsregistry.org/wiki/index.php/Part:BBa_K118002 BBa_K118002], [http://partsregistry.org/wiki/index.php/Part:BBa_K118003 BBa_K118003],[http://partsregistry.org/wiki/index.php/Part:BBa_K118008 BBa_K118008],[http://partsregistry.org/wiki/index.php/Part:BBa_K118001 BBa_K118001], [http://partsregistry.org/wiki/index.php/Part:BBa_K118004 BBa_K118004], [http://partsregistry.org/wiki/index.php/Part:BBa_K118007 BBa_K118007], [http://partsregistry.org/wiki/index.php/Part:BBa_K118014 BBa_K118014],[http://partsregistry.org/wiki/index.php/Part:BBa_I742158 BBa_I742158] . Plasmids were isolated from 10 mL overnight culture and analyzed by control restriction with enzymes EcoRI and PstI. Plasmid pBAD crtEBIY GFP was constructed from BioBrick [http://partsregistry.org/wiki/index.php/Part:BBa_K118005 BBa_K118005] and pBAD/araC fragment.

6/14/2010 –6/20/2010

BioBrick parts [http://partsregistry.org/wiki/index.php/Part:BBa_K274100 BBa_K274100],[http://partsregistry.org/wiki/index.php/Part:BBa_K274110 BBa_K274110] ,[http://partsregistry.org/wiki/index.php/Part: BBa_K274120 BBa_K274120],[http://partsregistry.org/wiki/index.php/Part: BBa_K274200 BBa_K274200] ,[http://partsregistry.org/wiki/index.php/Part: BBa_K274210 BBa_K274210] and [http://partsregistry.org/wiki/index.php/Part: BBa_K274220 BBa_K274220] were transformed into E. coli DH5α. Plasmids were isolated from 10 mL overnight culture and analyzed by control restriction with enzymes EcoRI and PstI. Cultures with carotene and lycopene operons were inoculated in 10 mL LB medium with added arabinose for induction of pBAD promoter. Cultures were incubated for 24 h at 37°C and 160 rpm. Β-carotene and lycopene were extracted from cells with acetone, using the procedure from Cambridge 2009 iGEM team. VIS-spectra (400-550 nm) were recorded, which confirmed the production of B-carotene, but not lycopene

6/21/2010 –6/27/2010

We tried to construct operon for zeaxanthin biosynthesis, using the existing biobricks for B-carotene synthesis and part I742158, but it appeared that all biobricks containing crtEBIY operon lacked SpeI site, which was later confirmed by sequencing.

Production media for B-carotene and lycopene production with increasing concentrations of arabinose to assess the responsiveness of pBAD promoter for different concentrations of arabinose.

6/28/2010 –7/4/2010

New procedure for B-carotene and lycopene production was tried out, using mixture of ethylacetate and ethanol (1:1 vol.), the 50°C heating step was omitted for the thermolability of carotenoids. Extracts were analysed using HPLC, which confirmed the production of predicted compounds. Primers for mutagenesis of SpeI site in B-carotene operone biobricks were designed and ordered.

7/5/2010 –7/11/2010

While waiting primers for the mutagenesis,  we tried to combine the construct by blunt-end ligation. We had problems with inactive restriction enzymes, so most of the cloning experiments were unsuccessful.

7/12/2010 –7/18/2010

Operones for B-carotene biosynthesis with SpeI site were amplified by PCR, but we were unable to ligate the long fragment into the vector.

7/19/2010 –7/25/2010

Same as last week.

8/2/2010 – 8/8/2010

Operon crtEBIY was reconstructed from crtEBI operon and crtY fragment with corrected SpeI site. That enabled us to construct crtEBIYZ operon for zeaxanthin biosynthesis. E. coli DH5α containing the plasmid with operon for zeaxanthin biosynthesis were inoculated to production media and incubated for 24 hours. Zeaxantin was extracted using the same protocol as previously for lycopene and B-carotene.

10/11/2010-10/17/2010

8/2/2010 – 8/8/2010

Operon crtEBIY was reconstructed from crtEBI operon and crtY fragment with corrected SpeI site. That enabled us to construct crtEBIYZ operon for zeaxanthin biosynthesis. E. coli DH5α containing the plasmid with operon for zeaxanthin biosynthesis were inoculated to production media and incubated for 24 hours. Zeaxantin was extracted using the same protocol as previously for lycopene and B-carotene.

8/9/2010 –8/15/2010

Production of zeaxanthin was confirmed by HPLC and compared to a standard.

8/16/2010 –8/22/2010

Fusion proteins with zinc fingers and genes for carotenoid biosynthesis were constructed, each one preceded by T7 promoter and followed by T7 terminator.

8/23/2010 –8/29/2010

We continued constructing above-mentioned fusion proteins.

8/30/2010 –9/5/2010

Results of sequencing  of fusion proteins revealed, that T7 promoter we were using was not functional. That was why we decided to change the strategy and started to build constructs RBS – ATG – zinc finger – enzyme under pBAD promoter.

9/13/2010 –9/19/2010

Sequencing of RBS-ATG-zinc finger-enzyme constructs showed that BioBrick parts with coding sequences for enzymes crtB, crtI and crtY ([http://partsregistry.org/wiki/index.php/Part:BBa_K118002 BBa_K118002] ,[http://partsregistry.org/wiki/index.php/Part:BBa_K118003 BBa_K118003] and [http://partsregistry.org/wiki/index.php/Part:BBa_K118004 BBa_K118004] , respectively) were not designed in such a way it would enable us to construct functional chimeric protein. A set of primers was designed to correct above-mentioned parts in a way which would allow us to construct fusion proteins.

We recieved plasmid with synthetic gene crtO.

9/20/2010 –9/26/2010

Genes crtB, crtI and crtY were PCRed, so we got constructs that allowed assembly of chimeric proteins. Constructs with RBS, zinc fingers and enzymes were assembled.

9/27/2010 –10/3/2010

PCR ligation of DNA program for biosynthesis was perfomed, PCR products were blunted with T4 DNA polymerase and ligated into pBluescript vector. Colony PCR on white colonies was performed and clones with the highest molar mass bend were sequenced.

Biosynthetic operons for carotenoid biosynthesis with enzymes liked to zinc fingers was beginning to be assembled. 

10/4/2010 – 10/10/2010

Constructs with pBAD promoter and first three or last three enzymes for carotenoid biosynthesis linked to zinc fingers were assembled.

Competent cells with plasmid pSB4C5 containing construct pBAD/AraC PSBII::crtY HivC::crtO Gli1::crtZ  were prepared.

10/11/2010 –10/17/2010

Above mentioned competent cells were transformed with plasmid pSB4K5 containing construct pBAD/AraC crtE::Jazz Blues::crtB crtI::Zif268. Competent cells from these transformants were prepared and transformed with pBluescript plasmids containing different programs for biosynthesis. As these cells were grown in media containing three different antibiotic the growth was very slow, which slowed down the testing of biosynthesis carotenoids.