Part:BBa_K4208003
PctD-LBD Expression
Contents
Usage
This composite part has been developed by team Tuebingen 2022 [1]to express the PctD-Ligand Binding Domain with 6xHis Tag in order to facilitate its purification via an Immobilized Metal Afffinity Column for poly HisTagged proteins. It is regulated by the lac promoter so that with the addition of IPTG to the growth media the cells start producing large amounts of protein. Later, this purified product can be used for experiments such an Isothermal Titration Calorimetry, as the one performed with PctD-LBD with Choline and Acetylcholine in our project. By exchanging the protein expressed in the device, other LBDs could be produced in order to characterize the interaction with ligands of interest.
To guarantee a high protein expression make sure:
- You use a high-copy number plasmid backbone - such as pET28a used in this case
- You use the correct concentration of IPTG as inducer - in our case 1 mM
Biology
PctD is a scarcely characterized chemoreceptor, found in Pseudomonas aeruginosa. It has been found that it binds to choline and acetylcholine. [2] We (iGEM Team Tuebingen) planned to express the PctD ligand binding domain (PctD-LBD) in BL21(DE3)pLys E.coli, purify it and use isothermal titration calorimetry (ITC) to check wether the PctD-LBD also binds the cyanotoxin Anatoxin-A.
We used a plasmid, that was kindly gifted to us by the lab of Victor Sourjik, that contained the His-tagged PctD-LBD to transform BL21(DE3)pLys E.coli cells and overexpress the protein.
To purify the protein we employed IMAC HisTrap column and the Äkta-FPLC system as described in [3]. We used SDS gel electrophoresis to analyze the expression and purification.
We attempted the ITC with the purified PctD-LBD and choline as a ligand. This was meant to be a positive control for our ITC experiment with Anatoxin-a as a ligand. In the end Anatoxin-a was not available for us to perform experiments with.
Results
In our project, we have induced a the production of PctD-LBD and purified the protein following the protocol from [4], available in an expanded version in[5].
SDS PAGE analysis suggested successfull expression of PctD-LBD protein after induction with IPTG. However, large parts of the protein were present in the unsoluble fraction after cell lysis. We successfully purified the soluble fraction of PctD-LBD protein. However, ITC experiments with choline suggested only 1 % of the protein was functional.
Based on these results, we advise other persons who want to use this part for protein expression with a pET-28a vector, to use the BL21-AI strain for expression, which had been used by the researchers for the initial characterization of PctD [6].
Sequence and Features
The sequence and features for this composite are found in this section, if you want more detail over the HisTag or the regulatory elements please visit the BioBrick pages set for them.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1147
Illegal XhoI site found at 2207 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1886
Illegal NgoMIV site found at 2241 - 1000COMPATIBLE WITH RFC[1000]
This plasmid was designed for [7] and it was a kind donation from Victor Sourjik's lab to provide us with it
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