Team:Groningen/Labwork/1 August 2013



Plates for the transformation of ΔDes, ΔCheY and ΔDes/ΔCheY did not show any colonies. The plates for eYFP showed some colonies on the plates with the transformation to E.coli. The plates with RFP and the ligation of Pdes/Chey did not show any colonies. Also the plates with a transformation of GFP240 and GFP0840 showed colonies.
Inoculated the colonies into LB with ampicillin to perform a miniprep later on. Miniprep analysis is done and the products are used to perform a restriction digestion. This showed that one of the eYFP colonies is a correct transformant. This transformant is plated on LB agar plates with IPTG and Amp resistance.
Made a new restriction digestion for RFP, eYFP and BBa_k823823 + LacI with EcoRI and PstI.
Inoculated B.subtilis colonies for transformation.
Made a restriction for Pdes with EcoRI and BamHI, for CheY with BamHI and PstI and for BBa_k823823 with EcoRI and PstI.
Heat inactivated the restriction digestions of Des Up, Tet, Des down, CheY up, spec, CheY down and Pdes.
Did a PCR clean up for CheY and BBa_k823823.
Did a ligation reaction for Des Up, Tet and Des down as well as a ligation reaction for CheY up, spec and CheY down. Heat inactivated these reactions and did a transformation to B.subtilis.
Made a ligation reaction for Pdes and CheY and heat inactivated the reaction. This ligation reaction is ligated into BBa_k823823. Also this ligation is heat inactivated and the reaction is transformed to E.coli.
Ligation reactions RFP and eYFP with BBa_k823823 + LacI are made and heat inactivated. Then they are transformed into E. coli.
Because the B.subtilis culture didn't grow that well, it is decided to plate a fresh stock on agar. If todays reaction fails. There is another chance tomorrow.


Made a miniprep of eYFP and RFP to get a higher concentration.
Did a gel purification to obtain purified fragments of eYFP, RFP and backbone BBa_k823823 + LacI.



Made a plasmid purification of GFP BBa_E0240 and got a concentration of 21,5 and also did a purification of GFP BBa_E0840 and got a concentration of 13,5.(placed in temp box labeled as GFP 0240 and GFP 0840)


The plates prepared yesterday show colonies. 4 colonies per plate are inoculated in LB + Ampicillin in order to isolate the plasmid on the day after and check for the presence of the desired insert.

To the tube, containing the purified BBa_K823023 + lacI digested with XbaI and PstI, 1µl SpeI is added and the tube is incubated for 1h at 37°C. After the treatment the backbone is ligated (3:1 insert:vector ratio) with BBa_E0840 and BBa_E0240 (already digested and purified yesterday with XbaI and PstI). The two ligation products are transformed into E. Coli DH5α and plated on LB + Ampicillin.

Bacillus Subtilis is inoculated in LB.

Inne & Sander

Did a inoculation of 2 colonies from the delta cheY plates and 2 colonies of the delta des Both plates were made yesterday.
Sample Strain Selection marker
1 Delta cheY 1 5 ug/ml cm
2 Delta cheY 2 cm 5 ug/ml
3 cheY negative control 5 ug/ml cm
4 Delta des 1 5 ug/ml cm 5 ug/ml km
5 Delta des 2 5 ug/ml cm 5 ug/ml km
6 Delta des negative control 5 ug/ml cm 5 ug/ml km