Team:UNICAMP-Brazil/Notebooks/October 1

MicroGuards

Preparation of electrocompetent E. coli

• Today we prepared more electrocompetent E. coli to use in our transformations according to Protocol 4.

Marcos and Taís

ColiGuard

Digestion Results

• Today we ran an agarose gel from the digested samples in order to assure that they were indeed digested. Unfourtunally, only BBa_I718017 and BBa_I718016 (Lox parts) were sucessful digested (vector linearized). Biobricks BBa_J61000 and BBa_E0840 appeared in the gel as a single band, reaching the approximately size of the backbone vector. No fragment excised by digestion =(
• We repeated the digestion procedure for the biobricks that didn't work and, once more, we couldn't digest them!
• We believe that those biobricks came without the insert! Only backbone vector is available...
• Without those parts, it turns a lot more difficult to assemble our necessary devices =/

Marcelo

Kamikaze System Assembling Begins!

• We did 2 inoculum with 4ml of E. coli with the plasmids BBa I746911 and BBa K112806 that have just arrived. We were waiting for then to start the Kamikaze System! .

Ane

Cre-Recombinase Digestion

• Once we have purified samples for Cre-Recombinase and for the vector pSB1A3, we could proceed on digesting both of them with XbaI and SpeI restriction enzymes.
• Digestion lasted 3 hours on both cases.

Víctor

YeastGuard

New biobricks - screening by miniprep and digestion

• Today we did the miniprep using the rest of the transformed E. coli that was not plated in LB+AMP media. After that we digested the miniprep with Xba1, in order to find the band with the correct molecular weight (expected for the inserts connected to the vector). Unfortunately we only found one band with the wrong molecular weight.

JEN1 ORF

• The concentration of the new part JEN1ORF wasn´t enough for the ligation reactions. So we repeated the PCR and today we digested it with Xba1 and Spe1.

Terminator Biobrick

• We digested the terminator briobrick PCR product with Xba1 and Pst1.

Taís