Team:Groningen/Labwork/31 July 2013



Examined the plates of eYFP and GFP grown on Lb agar + cm plates. This reveals that there are cells growing and the negative control did not show any colonies. Six colonies per type are inoculated in LB medium with cm to perform a mini prep later on.
Examined the plates of the ligation product BBa_k823823+LacI with eYFP and GFP. These plates did not show any colonies, although the negative control did, which is very strange.
The plated B.subtilis strain did grow. So three single colonies are inoculated to perform a transformation reaction.
Made a restriction digestion for RFP and eYFP (EcoRI and PstI).
Did a mini prep for the inoculated samples of RFP and eYFP. The concentration is high enough to store the samples for restriction digestions.

Chaline & Mirjam

Made a restriction digestion for Pdes (BamHI) and BBa_k823823 + LacI (EcoRI and PstI).
Did a mini prep to obtain more BBa_k823823 + LacI. Made a restriction digestion of these samples to verify the plasmid is correct. Did a nanodrop to measure the concentration of the samples. The samples are fine, with concentrations between 30 and 60 ng/µl.
Run a 0.8% agarose gel on the samples of the restriction of the backbone to do gel purification. Gel purified with a very low concentration of the backbone. Though it is decided to continue with this sample.
Did a ligation for Pdes and CheY. Did a restriction on the ligation with EcoRI and PstI.
Did a ligation reaction for ΔCheY.
Did gel extraction for RFP and eYFP. These samples have a very low concentration.
Made a restriction digestion for RFP and eYFP. This revealed high concentrations for both and a ligation into the backbone is made.
The leftover ligation reaction of the backbone with RFP/eYFP of 30-07 are transformed to E. coli.
The ligation reaction of pDES and CheY is transformed to E. coli
The ligation reactions of ΔCheY, ΔDes and a combination of both are transformed to B.subtilis


Made a plasmid purification of Munchen backbone with Lacl. Got a concentration of 44,4 and 47,3 ng/ul. (placed in temp box labeled as Mun+Lacl)
Made a spread of the Des knockout on LB agare with Cm 5 ug/ml and Kn 5ug /ml. Left to grow in 37°C.
Made a spread of the CheY knockout on LB agare with Cm 5 ug/ml. Left to grow in 37°C.


BBa_K823023 + lacI, BBa_E0840, BBa_E0240 and mKate2 are digested with XbaI and PstI.
The samples are checked on agarose gel 0.8%: the digestion worked (Friso).
The samples are purified using the PCR purification kit.
BBa_K823023 + lacI is ligated to BBa_E0840, BBa_E0240 and mKate2 (1:1 insert:vector ratio).
The ligation products are transformed to E. Coli DH5α and the competent cells are plated on LB + Amp.

BBa_E0840 and BBa_E0240 are transformed again and grown in LB + Chloramphenicol.

LB agar aimed to grow Bacillus Subtilis biofilm is prepared.