*<p style=”text-align:justify;”>Since yesterday's digestion didn't work, we repeated today the digestion procedure for Cre-Recombinase's biobrick. We used the same restriction enzymes, but letting the digestion last 5 hours instead of 3.</p>
+
*<p style=”text-align:justify;”>Then, we ran an agarose gel, in order to assure that digestion indeed happened. Unfortunately, it didn't =/</p>
+
*<p style=”text-align:justify;”>After performing a few tests, we realized today that our ''Spe''I restriction enzyme was no longer capable of performing its activity.
Although we had experienced a low efficiency in biofusion usage, we won't continue finO's biobrick construction.
Marcelo
finOP - Final Conclusion
After several unsuccessful attempts on constructing finOP's biobricks, using different strategies with different backbones, our team decided to leave aside those biobricks.
Finalizing these parts will demand a lot of time, and we intend to already send our assembled biobricks this Thursday.
It's quite possible to fully assemble them, but we haven't enough time right now. We got a lot of work to do related on characterizing our assembled biobricks and updating this wiki you are currently reading on.
Thus, we will now focus on other kind of work. We are done with finOP inhibition system.
Marcelo
The BBa_B0014-BBa_K112806 + BBa_I746911 ligation and transformation
Today looks like a good day. We made 4 Miniprep (Protocol 2) for the colonies selected. We made the DNA quantification and then we made the digestion with enzymes restriction XbaI and PstI. Likewise, we made the digestion of BBa_I746911 with enzyme XbaI and PstI. We made the ligation reaction to join the new part BBa_K112806 and the terminator BBa_B0015 with the plasmid part BBa_I746911. After the dyalisis, we improved the transformation. We expect to get finally a BioBrick.
Luige & Ane
PY Promoter - Colony-PCR screening
We selected 3 colonies of each transformation we did yesterday to perform a colony-PCR screening. We used 2 pairs of primers and analyzed the results of our PCR in an agarose gel:
- VF/VR, the verification primers of BBa_J23100 and BBa_B0015 plasmids:
- Ppy-F-1 and Ppy-R, the pair of primers that amplify PY1 promoter:
The expected size for PY1 + RFP + BBa_J23100 amplified with VF/VR primers is 1201 bp.
The expected size for PY1 + RFP + BBa_B0015 amplified with VF/VR primers is 1279 bp.
The expected size for PY1 + RFP + BBa_J23100 and PY1 + RFP + BBa_B0015 amplified with PY primers is 133 bp.
As we can observe in the gel photo, all the colonies presented bands compatible with the size expected for both pairs of primers.
So we inoculated 3 colonies in liquid LB-AMP (for BBa_J23100) and 3 colonies in LB-AMP-KAN (for BBa_B0015) at 37ºC to perform mini-preps for plasmid extraction tomorrow.
Fabi and Léo
Cre-Recombinase without ATG + terminator
Since yesterday's digestion didn't work, we repeated today the digestion procedure for Cre-Recombinase's biobrick. We used the same restriction enzymes, but letting the digestion last 5 hours instead of 3.
Then, we ran an agarose gel, in order to assure that digestion indeed happened. Unfortunately, it didn't =/
After performing a few tests, we realized today that our SpeI restriction enzyme was no longer capable of performing its activity.
<p style=”text-align:justify;”>The Lysozyme+biofusion2 plate grew! We screened the plate by colony PCR and found two expected fragments (nº 2 and 9). We did miniprep of these colonies.
We did the colony PCR to find positive colonies for the other 2 final biobricks: pJEN1 and pDLD. We found 8 positive colonies of pDLD+biofusion2 =) and 3 positive ones of pJEN1+biofusion2.
We transformed Adh1+lysozyme+Biofusion in competent E. coliand plated in LB+Amp media.
We did miniprep of 3 colonies of JENorf-pGEM, digested it with EcoRI and SpeI in order to release the part with the EcoRI site and to connect it in biofusion1. We found no expected fragments (1873bp). We had to decided to give up on it, because it is still on pGEM and there are two more clonnings to reach the biobrick format. We are going to focus our energy on the most promising biobricks right now.
pADH1+YFP
Today we made the miniprep and the digestion of the plasmids containing our new briobrick (pADH1+YFP). The enzymes used were XbaI and PstI.
Then, we performed the purification of the bands corresponding to the size of this new biobrick pADH1+YFP. So, it is going to be sent to IGEM organization. =)
Wesley and Sula
YEP
Today we did the miniprep (Protocol 2) and we digested YEP358 – β-galactosidase plasmid with XbaI/SpeI.
"
