*<p style=”text-align:justify;”>Today, we tried to join our CeiB part into the plasmid Biofusion. We made the enzyme restriction, nevertheless we had serious problems with the agarose gel and we lose the samples.
A lot of colonies have grown with the reaction with only BBa B0015 and in the reaction with BBa B0015 + BBa K112806, we selected 15 to do a Colony PCR, none of them was positive. These results show that coudn't do a good digestion of BBa B0015, so tha's why it's recircularizing and giving a lot of fake positives.
To improve our digestion we let BBa B0015 digesting ON with EcoRI and tomorrow we will purify it from agarose gel to make sure we only get linearized plasmid, then we will digest with XbaI tomorrow.
Marcos
PY Promoter - Digestion and ligation reactions
Today we digested PY1 + pGEM with EcoRI and SpeI to excise PY fragment from pGEM vector.
We also digested the plasmid BBa_J23100 (that one with the RFP reporter) with EcoRI and SpeI. With this plasmid digested with this 2 enzymes it is going to be possible to insert our digested PY fragment into it.
These digestion reactions lasted for 3 hours.
After the digestion we performed the ligation reaction of PY1 + BBa_J23100 following Protocol 11.
Fabi and Léo
CeiB: Problems with agarose gel
Today, we tried to join our CeiB part into the plasmid Biofusion. We made the enzyme restriction, nevertheless we had serious problems with the agarose gel and we lose the samples.
Ane e Luige
YeastGuard
New strategy: pGEM
<p style=”text-align:justify;”>We confirmed the insertion of pJEN1 in pGEM by colony PCR using the part's internal forward primer and the vector's reverse primer (M13). Unfortunately there were no transformed colonies with JENorf, neither lysozyme.
We continued screening JENorf and Lysozyme! To find positive colonies we screened 10 more colonies of each plate and made a backup inoculum in case we find right ones. We found 7 positive colonies for lysozyme, unfortunately no positive colonies were found for JENorf again.
YFP+Terminator
We did colony PCR to amplify the fragment between the vector's annealing sites (vector's primers forward and reverse) that we expected to be the YFP linked to the terminator sequence. We found some strange fragments in YFP-End PCR. This fragaments correspond to the vectors size (YFP Biobrick) without the insert (End). We believe that this fragment correspond to the recircularized vector. Our expected fragment wouldn't be amplified, since it has 3000pb, so we decided to perform miniprep from the colonies that showed no amplicons! We hope we are right! =)
Raíssa
YEP358: yeast expression plasmid
We decided to use this vector to test our constructions in expression experiments with S. cerevisiae. This plasmid has a polylinker site that permits to insert our constructions. Fortunately it has XbaI and PstI sites that make the clonning easier for us. In adition, the vector contains an Ura3 gene, used to select transformed colonies since the medium used have no Uracila. Because of our problem with the terminator biobrick we decided to cut of part of the plasmid's β-galactosidase gene and use it´s terminator.
"
