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Speed Control - Methodology

CheZ knockout of YBE01 and YBS01

Recombineering strategy is used for the cheZ gene of YBE01 and YBS01. Because the protocols for the knockout process are the same for these two strains, we just took YBS01 as an example to illustrate the whole procedures.

Step 1

A psim6 plasmid, which could help Recombineering process, was transformed into the competent cell of YBS01 by electroporation. After recovering with SOC solution for 30 minutes, they were spread to Agar plates with Ampicillin resistance. Then one single colony on the plate was picked up for pre-culture.

Step 2

A DNA fragment with homologeous arms made by PCR using specific primers was transformed into the YBS01 with psim6. After recovering with SOC solution for 30 minutes, they were spread to Agar plates with Chloramphenicol resistance.

Step 3

PCR was done with test primers and with single colonies on the plate as templates to examine if the recombination strategy was successful.

Regulation of cheZ expression

Step 1

MG3 (delta CheZ) strain was used to test whether the inducible regulation of cheZ would succeed. With no background expression of cheZ, we would be able to achieve precise control over intracellular cheZ level through regulated gene expression. The plasmid pIac-his-cheZ-cm was transformed into the competent cell of MG3 and different cheZ expression level was induced through incubating with different concentrations of IPTG and various induced time. The samples were gathered at specific time and with different IPTG concentrations as follows.

Step 2

The bacteria bacteria lysis underwent to release the protein samples. BCA quantification analysis was done to detect the concentrations of these protein samples. After analyzing the concentrations of each protein samples, we used the formula: total amount of protein= sample concentration*sample volume to adjust the sample volume to the same loading volume by adding the mixture buffer. Equal loading of protein samples were ensured in Western blotting after these adjustments.

Step 3

A wider range of IPTG concentrations were added to MG3 bacteria with the plasmid plac-his-cheZ-cm.