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A single-protein calcium ion sensor mediating cell survival

iGEM team Freiburg

Introduction

Split enzymes:
Some enzymes can be divided into two separate parts that won´t show any activity until both parts are physically brought together again;
this circumstance allows enzyme-activity-assays.
As those assays were already being used in our lab before, we could access plasmids containing enzyme-halfs of beta-lactamase and Dihydrofolatereductase (DHFR).

Calmodulin:
This molecule is known for its strong conformational change upon binding calcine.
Ikura, Truong et al.(Ontario Cancer Institute and Department of medical Biophysics, University of Toronto) have fused CFP and YFP to the ends of modified calmodulin and shown that you can then induce FRET between them by adding calcine.
This work inspired us to test calmodulin as "switch" for our split enzymes. Prof. Ikura was so friendly to send us his calmoduline (as plasmid with sequence, thanks a lot again!) so that we could gain it via PCR.

The idea:
Was to attach those enzyme-halfs to several trigger-proteins (here: calmodulin) by cloning each one of the enzyme-halfs and the trigger-protein together in one plasmid.
This plasmid would then contain the code for the whole "protein-machinery", allowing in vivo-tests as well as expression and purification of the engineered protein, an on/off-switchable enzyme.

Picture: DHFR1(pink)-Calmodulin(green)-DHFR2(blue)-Construct, active state; calcine bound

Picture: DHFR1(pink)-Calmodulin(green)-DHFR2(blue)-Construct, inactive state; no calcine bound

Materials and Methods

Results

Plasmids:
We gained plasmids containing both parts of DHFR linked to calmoduline by 2/4/6 glycins; in-vivo-growth tests showed the best activity for 4-glycine linkers;
thus we decided to proceed with this linker-length.

In-Vivo-Analysis:
As DHFR is essential for bacterial growth in minimal media with trimethioprim (TMP), we could show that the ability of E.colis with our plasmid to grow is directly dependant on the presence -and, furthermore, concentration- of calcine-ions (Ca2+)in the media:

Discussion

References

split DHFR

Pelletier JN, Arndt KM, Plückthun A, Michnick SW. "An in vivo library-versus-library selection of optimized protein-protein interactions." Nat Biotechnol. 1999 Jul;17(7):683-90.

Pelletier JN, Campbell-Valois FX, Michnick SW. "Oligomerization domain-directed reassembly of active dihydrofolate reductase from rationally designed fragments." Proc Natl Acad Sci U S A. 1998 Oct 13;95(21):12141-6. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=9770453&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum pubmed]

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