pKUC3

Protein Expression Prokaryotic cells - B. licheniformis PphyL′-′amyE fusion

Experiment
Protein Expression Prokaryotic cells - B. licheniformis PphyL′-′amyE fusion
Product
pKUC3 from Britta Jürgen, Pharmaceutical Biotechnology, Institute of Pharm
Manufacturer
Britta Jürgen, Pharmaceutical Biotechnology, Institute of Pharm

Protocol tips

Protocol tips
The vectors were used to transform the B. licheniformis strain MW3 by electroporation [20] resulting in the strains TH3 (pKUC3) and TH4 (pKUC4), respectively. These strains were then cultivated in phosphate-limited BMM as already described.
Downstream tips
Culture supernatants for enzyme assays were taken at different time points during the cultivation. The first sample was taken during the exponential growth phase (four hours after cultivation), the second sample during the transient phase and additional samples were taken 2, 4, 6, 8, 10, 12 and 14 h after onset of the stationary growth phase.

Publication protocol

The activity of the PphyL promoter was analyzed by means of translational reporter gene fusions. For this purpose, an approximately 300-bp fragment containing the PphyL promoter from B. licheniformis DSM13 (Additional file 1: Figure S2) was cloned in front of the α-amylase and xylanase reporter genes from B. subtilis 168 with the primer pairs 1/2 and 1/5, respectively (Additional file 1: Table S1). The amyE and xynA genes from B. subtilis 168 were amplified with the primer pairs 3/4 and 6/7, respectively (Additional file 1: Table S1). The PphyL′-′amyE and PphyL′-′xynA fusions were constructed by means of the precise gene fusion polymerase chain reaction strategy described by Yon and Fried [18] by using the primer pairs 1/4 and 1/7, respectively. The PCR-fusion fragments were then inserted into the XbaI and KpnI sites of the multi-copy plasmid pKUC (this shuttle vector is based on the pUC18 and pKTH290 plasmids [19]) resulting in vector pKUC3 and pKUC4, respectively. These vectors were used to transform the B. licheniformis strain MW3 by electroporation [20] resulting in the strains TH3 (pKUC3) and TH4 (pKUC4), respectively. These strains were then cultivated in phosphate-limited BMM as already described. Culture supernatants for enzyme assays were taken at different time points during the cultivation. The first sample was taken during the exponential growth phase (four hours after cultivation), the second sample during the transient phase and additional samples were taken 2, 4, 6, 8, 10, 12 and 14 h after onset of the stationary growth phase.

Full paper   Login or join for free to view the full paper.

Reviews

pKUC3 from Britta Jürgen, Pharmaceutical Biotechnology, Institute of Pharm has not yet been reviewed for this experiment

We'd love it if you would be the first to write a review!

Discussion

Start your discussion

Share your thoughts or question with experts in your field

Start a discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Protein Expression Prokaryotic cells - B. licheniformis PphyL′-′amyE fusion using pKUC3 from Britta Jürgen, Pharmaceutical Biotechnology, Institute of Pharm.

View full paper   Login or join for free to view the full paper.

Manufacturer protocol

Download the product protocol from Britta Jürgen, Pharmaceutical Biotechnology, Institute of Pharm for pKUC3 below.

We haven't found the manufacturer protocol for this product yet.

Videos

Check out videos that might be relevant for performing Protein Expression Prokaryotic cells - B. licheniformis PphyL′-′amyE fusion using pKUC3 from Britta Jürgen, Pharmaceutical Biotechnology, Institute of Pharm. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.

We haven't found any additional videos for this experiment / product combination yet.

Outsource your experiment

Fill out your contact details and receive price quotes in your Inbox

  Outsource experiment
Become shareholder Discussions About us Contact Privacy Terms