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Team:Slovenia/METHODS and PARTS/notebook/viola
From 2010.igem.org
week 1
week 2
week 3
week 4
week 5
week 6
week 7
week 8
week 9
week 10
week 11
8/9/2010-8/15/2010
DH5α cells were transformed with three plasmids (taken from the Registry) containing the violacein operon. (BBa_K274002, BBa_K274003,BBa_K274004). The cells were grown on LB Kan/Tc agar plates. Transformation of part Bba_K274002 was unsuccessful – there were no colonies on the LB Tc plates. Colonies containing plasmids with parts BBa_K274003 and BBa_K274004 were later inoculated into mini-prep flasks with Kan. Plasmid DNA was isolated from the over-night cultures.
Primers for amplificiation of separate vio genes were designed and ordered, and a table of constructs, necessary for the experiment was made.
8/16/2010-8/22/2010
Primers arrived and PCR was performed. The reaction was successful for all vio genes, except for the vioC gene. We later discovered some secondary structures of the reverse primer, so the primer was modified and re-ordered. The fragments were isolated from the gel, cut with EcoRI and SpeI enzymes and ligated into the previously cut pSB1AK3 vector containing the T7 terminator. DH5α cells were transformed with the ligation products and put on LB Amp agar plates. Single colonies were inoculated into mini-prep flasks with Amp and plasmid DNA was isolated from the over-night cultures. Restriction analysis was performed with EcoRI and PstI enzymes.
8/23/2010-8/29/2010
Primers for the T7 promotor arrived and primer annealing was performed. The fragment was ligated into the previously cut pSB1AK3 vector and DH5α cells were transformed with the ligation products and put on LB Amp agar plates. Single colonies were inoculated into mini-prep flasks with Amp. Plasmid DNA was isolated from the over-night cultures and sent to sequencing.
The pSB1AK3 vector containing the T7 promotor was cut with SpeI and PstI enzymes and isolated from the gel. Zinc-finger fragments were ligated into the vector and DH5α cells were transformed with the ligation products and put on LB Amp agar plates. Single colonies were inoculated into mini-prep flasks with Amp. Plasmid DNA was isolated from the over-night cultures.
8/30/2010-9/5/2010
We got the sequencing results, and apparently something went terribly wrong with the T7 promotor, so we decided to change our plans - the vio genes were now to be expressed under the pBAD promotor. The PCR for amplification of vio genes was performed again, the fragments were isolated from the gel and cut with XbaI and PstI enzymes. They were later ligated into the previously cut pSB1AK3 plasmid containing RBS plus corresponding Zinc-finger sequences. DH5α cells were transformed with the ligation products and put on LB Amp agar plates. Single colonies were inoculated into mini-prep flasks with Amp and plasmid DNA was isolated from the over-night cultures.
9/6/2010-9/12/2010
Finally, the PCR for amplification of vioC was successful. The amplified fragment was isolated from the gel and cut with XbaI and PstI enzymes. The resulting DNA fragment was ligated into the pSB1AK3 vector containing RBS, and into the pSB1AK3 vector containg RBS plus the corresponding Zinc-finger. DH5α cells were transformed with the ligation products and put on LB Amp agar plates. Single colonies were inoculated into mini-prep flasks with Amp and plasmid DNA was isolated from the over-night cultures.
9/13/2010-9/20/2010
The constructs of fusion proteins (Zinc-finger – linker – enzyme) were cut in such a way, that they could be ligated together. The Zinc-finger – vioA construct was ligated with the Zinc-finger – vioB construct and the Zinc-finger – vioE construct was ligated with the Zinc finger – vioD construct. DH5α cells were transformed with the ligation products and put on LB Amp agar plates. Single colonies were inoculated into mini-prep flasks with Amp and plasmid DNA was isolated from the over-night cultures.
The plasmid containing part BBa_K274004 (vioABDE) was cut with SpeI and PstI enzymes and ligated with the previously cut vioC fragment with RBS. DH5α cells were transformed with the ligation products and put on LB Kan agar plates. Single colonies were inoculated into mini-prep flasks with Kan.
9/21/2010-9/26/2010
The Zinc-finger – vioE – Zinc-finger – vioD construct was ligated with the Zinc-finger – vioC construct. DH5α cells were transformed with the ligation products and put on LB Amp agar plates. Single colonies were inoculated into mini-prep flasks with Amp and plasmid DNA was isolated from the over-night cultures.
Zinc-finger – vioA – Zinc-finger – vioB and Zinc-finger – vioC – Zinc-finger – vioD – Zinc-finger – vioE constructs were cut with XbaI and NotI restriction enzymes and ligated into the previously cut pBAD_BsaI – DTER construct, each in a different vector. The first construct was inserted into a pBAD_BsaI – DTER construct in a vector with Cm resistance, and the second construct into a pBAD_BsaI – DTER construct in a vector with Kan resistance. DH5α cells were transformed with the ligation products and put on LB Cm/Kan agar plates. Single colonies were inoculated into mini-prep flasks with Cm/Kan and plasmid DNA was isolated from the over-night cultures. Restriction analysis was performed on the isolated plasmids.
9/27/2010-9/3/2010
Cells containing constructs vioABCE and vioABCDE were inoculated into mini-prep flasks with Kan. Violacein and deoxychromoviridans were extracted from the over-night culture and the absorbance spectra of the resulting samples were measured.
10/4/2010-10/10/2010
Competent cells carrying plasmid with PBAD-Zinc-finger-vioA- Zinc-finger-vioB-DTER construct were made. These cells were than transformed with construct PBAD-Zinc-finger-vioe-zinc-finger-viod-zinc-finger-vioc-dter and put on LB Kan ,Cm plates. Single colonies were inoculated into mini-prep flasks with Cm/Kan. Over night culture was than put in 100 ml LB medium. This culture was than used to make competent cells carrying 2 plasmids: one with PBAD-Zinc-finger-vioa-zinc-finger-viob-DTER construct (cloramfenikol resistance) and one with PBAD-Zinc-finger-vioe-zinc-finger-vioc-zinc-finger-viod-DTER construct (kanamicin resistance).
10/11/2010-10/17/2010
Competent cells from week 9 were transformed with plasmids carrying a DNA program, a scrambled DNA program and a pBluescript plasmid (without a DNA program). Overnight cultures were diluted in fresh LB medium to an optical density of 0,05 and grown at 30°C in the presence of Amp, Kan and Cm. Samples were taken every hour of the incubation. No change in color was observed, so the cultures were incubated over night and gained purple color by the morning.
10/18/2010-10/24/2010
The experiment from week 10 was repeated. We began taking samples after 8 hours of incubation and repeated the procedure at various times for the next 37 hours. Violacein was extracted from individual samples with ethyl acetate. Extracts were analyzed by TLC and quantitative determination of violacein was performed by denzitometry at 575 nm after scanning the spectra directly on the HPTLC plates. Violacein was also identified by mass spectrometry. The quantity of violacein production with or without nucleic acid program or in the presence of scrambled nucleic acid program was compared.
