Team:Groningen/Labwork/28 September 2013



The CheC plate does not show any colonies. So a new restriction digest is made for CheC up, Spec and CheC down. These restrictions are heat inactivated and a ligation in a 1:1 ratio is made. Again the reaction is heat inactivated. The ligation product is transformed into the B. subtilis ΔCheYΔDes strain.

A mini prep analysis is done for EstA-S3 and EstA-S9. After which a restriction digestion is done with E-S. A restriction digestion for S5 and S11 is made with X-P. A ligation is made between Esta-S3 and S5, and Esta-S9 and S11. These ligation products are ligated with the submission backbone and transformed into E. coli.

A gel purification is done for Pdes-CheY and BBa_K823023. However the concentration was to low to work with. So a new restriction digestion is made for Pdes-CheY and the integrational backbone. These parts are ligated together and transformed to E. coli
To verify if Pdes-CheY is working. The construct is restricted with EcoRI and SpeI, and ligated to the restricted GFP-DSM (XbaI-PstI). This ligation product is transformed into E. coli.

Did a colony PCR to check if the mutant strains still contain the ΔCheY and ΔDes, but no conclusive results came up. Because the strains are still growing, it is chosen to do one more purification round. As a control the wild type strain is plated on the same antibiotics to see the difference in growth.