Asaia

Ligation/Transformation C3+Promotor(weak/strong)+RBS Ligation/Transformation C3+Promotor(w/s)+RBS+Immuno

Today we transform competent Asaia with a GFP + kanamycin (resistance) following this protocols. We plate it and we'll waiting 2 days to see if it works. we do digestion to create our first biobrick. We will make the ligation and the transformation in E-Coli tomorrow.

BioBricks

Digestion/Ligation of ORI + Resistance (Amp/Kan) into Base Vectors

• Made some plates with GLY medium and Agar.

we count the drosophile and take photos with the fluorecense microscope. In some tubes the filter has detached so the expermiment for those tube are finished.

Immunotoxin

We have designed primers that overlap, generating a promoter and ribosome binding site. We want to put this in a vector which would be our basis to add other genes, for example our immunotoxin. We designed a strong promoter+RBS and a weak one, just in case the strong one generates too much immunotoxin and kills the bacteria...

Wiki

We did a lot of work on the wiki but we haven't downloaded it yet.

Asaia

We keep counting the drosophila and taking pictures with the GFP filter.

Biobrick

• Digestion of the plasmid containing the origin + one resitance (either Amp, Kan or Ch) and digestion of the vector i51020
• Ligation ofthe the different parts (one insert in one back bone vector) => origin+resistance+backbone_vector_for_biobrick
• Plating over night => we obtain something on the plate for the ori + amp nothing on the other

Immunotoxin

Henrike verified the primer sequences for us and ordered them --> they should arrive on Friday or Monday.

Asaia

• The asaia transformation we let in the incubator since 02-08 are now showing colonies! As expected and verified under the fluorescent microscope, they express GFP.

• Transformed Asaia with the pB129421248??? plasmid from E. coli to see whether it works in Asaia.

Other

• Continued counting the dead Drosophilae
• Made 2l of liquid gly medium
• Put 2 5ml cultures of recovered wt-Asaia in the incubator to make competent cells

Biobrick

• Make 5 ml miniprep to verify the ori + ch + i5010
• Make negativ control for the ori + ch + i5010 on Lb+Amp plate (as the ori + amp were in the A3 backbonevector)

Other

• Continued counting the dead Drosophilae and taking pictures.
• Measured the OD of the two 5ml wt-Asaia cultures (4.1 and 4.7),diluted the cultures to obtain a solution with OD 0.6 tomorrow morning and finally make competent cells

Biobrick

• The previous ligation didn't work at all, so we made a new ligation with ori + res + i51020
• We replated the collonies that had well grown on the plate the 28.7 to keep them in glycerol

Asaia

• We made a culture to make them competent toworrow (06.08)

Biobrick

• We obtained colonies on the plate from yesterday (Asaia origin + Resistance). We made a miniprep from that and stored some on Glycerol in the -80°C freezer. With the same miniprep we run a gel. Result :

Asaia

• Measured the OD of our Asaia cultures. They were 1.24 and 1.95. We diluted them again and then started the procedure to make competent cells. Finally we got 22 aliquots of 150ul.
• Had fun with the liquid nitrogen left over...
• Plated one aliquot on Gly without AB to make sure the now hopefully competent cells survived the procedures.
• We made some stock of "asaia origine + Resistance (Kan, Cm, Amp) + BackBones Vector" in the -80°C fridge.
• We've transformed Asaia with plasmid C3 + E-coli Origin + Resistance and the colonie didn't grow so we can say it out and loud :

E-coli Origin doesn't work in Asaia