Team:Brown/Parts

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BioBricks

<groupparts>iGEM010 Brown</groupparts>

Part:BBa_K324000 - His-tagged LacI repressor protein in RFC25 format

This part consists of the lacI repressor protein (BBa_I732100) optimized with endings for protein fusion in the Freiburg assembly format (RFC25). There is also a 6-his tag appended to the C-terminus for easy purification using Ni-affinity column. RFC 25 prefix/suffix and 6-his sequences were attached via PCR.

Design Notes

6-his tag was appended to the 3' end of this protein to allow for purification upon expression in E. coli. We chose a C-terminal tag to avoid isolation of incompletely translated peptides.

Source

[http://partsregistry.org/Part:BBa_I732100] Submitted by the USTC '07 iGEM team, taken from the 2010 Spring Distribution

Part:BBa_K324001 - His-tagged AraC regulatory protein in RFC25 format

This part consists of the AraC regulatory protein optimized with endings for protein fusion in the Freiburg assembly format (RFC25). There is also a 6-his tag appended to the C-terminus for easy purification using Ni-affinity column. The LVA rapid degradation tag from our source DNA, BBa_C0080, was also removed from the C-terminus. All modifications were made to BBa_C0080 by means of PCR with specially designed primer set.

Design Notes

The LVA tag was removed because this part's intended purpose, harvesting from cells and purification, required the protein to have a long lifespan. 6-his tag was added to the C-terminus to avoid isolation of incompletely translated peptides during Ni-affinity purification.

Source

refer to [http://partsregistry.org/Part:BBa_C0080]

Part:BBa_K324002 - Tat protein transduction domain with glycine linker (RFC25)

This part consists of the Tat protein transduction domain, an 11 a.a. sequence able to carry attached proteins across cell membranes without need to permeabilize. This domain, attached to a glycine linker, can be joined to other peptides in the RFC25 Freiburg assembly format to create fusion proteins that can freely transduce cell membranes. Our part consists of the Tat-glycine linker, intended to be attached to the N-terminus of any fusion protein construct.

The original Tat a.a. sequence comes from the Tat protein in human immunodeficiency virus but does not possess any innately pathogenic features. Bsl2 safety precautions are generally recommended for Tat-fusion proteins, although Tat DNA can be manipulated without special protocols.

Design Notes

The RFC25 fusion assembly format was used because this protein domain is intended to be fused to other proteins to convey its membrane-crossing characteristics. N terminal and/or C terminal addition of the Tat domain have both been shown to be effective.

Source

We originally obtained the Tat-PTD DNA from Will Donovan (william_donovan@brown.edu) as part of the Tat-PTD_ScFV project.

Part:BBa_K324003 - TetR repressible generator of LacI

This part combines two biobricks, a TetR repressible promoter+RBS (BBa_J13002) and the LacI regulatory protein in RFC25 assembly (BBa_K324000). This BioBrick allows the expression of LacI when inserted into E. coli, for the purpose of testing constructs controlled by the LacI promoter.

Design Notes

These parts were joined together with standard biobrick assembly, even though the LacI protein was in RFC25 and thus capable of protein fusion assembly as well.

Source

A simple Biobrick assembly was conducted to join the TetR promoter+RBS ([http://partsregistry.org/Part:BBa_J13002 BBa_J13002] with LacI [http://partsregistry.org/Part:BBa_K324000 BBa_K324000]. J13002 was obtained from the 2010 Spring Distribution, while K324000 was adapted by 2010 Brown iGEM from an existing part. See the part's page for further details