Team:Brown/Parts

From 2010.igem.org

Parts

Biobricks sent.JPG

Contents



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

[1] 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 [2]


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 (BBa_J13002) with LacI (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


Part:BBa_K324004 - AraC inducible ChFP generator

This part combines two previously available BioBricks via standard assembly: a promoter inducible by AraC (BBa_R0080) and CherryFP generator (BBa_J06702). This composite acts a reporter in E. coli, allowing cells to produce cherry fluorescent protein in the presence of AraC protein. In the absence of AraC, minimal levels of fluorescence should be observed.

The cherry FP generator consists of an RBS, mCherry, and double terminator.

Source

Both of this composite's constituent parts, the AraC inducible promoter (BBa_R0080) and CherryFP generator (BBa_J06702), are available from the Spring 2010 Distribution.


Part:BBa_K324050 - TrpR-RBS-TetR-Term-pTetR-RBS-CI-RBS-AraC-RBS-Mnt-Term

This part is a double repressor system derived from Part:BBa_K191005. A Trp promotor controls expression of tetR, a repressor to the Tet promotor. The Trp promotor is ON, unless otherwise repressed. The expression of CI, AraC, and Mnt, which follow the Tet promotor, is inhibited given the absence of tryptophan.

Design Notes

Because there are illegal SpeI sites in the Trp promotor, all assembly had to be done with RFC21 standard (bgl bricks). Thus, we had to add the appropriate bgl-brick sites to all parts we worked with. See our team page for a more in depth description.

Source

Part:BBa_K191005 for TrpR-RBS-TetR-Term-pTetR (PCR to isolate this segment) Part:BBa_B0034 for sequence of RBS (added RBS by PCR to CI, AraC, and Mnt)


Part:BBa_K324051 - LacI-RFP-Bistable part-GFP

This is the Bistable part originally created by team PKU '07, modified by PKU '09, and now further modified by us. We have added expression of LacI before RFP and the CI434 regulated promotor. As described in , and detailed here this part has two stable states. Initially, the green, or CI434 state dominates. However, as CI is introduced, expression of CI434 (and thus state 1) is inhibited, and the bistable part switches to state 2, where CI and RFP are produced. We have added the additional expression of LacI in state 2.

Design Notes

See our project page for detailed design considerations.

Source

BBa_K2280034 for Bistable part, BBa_C0021 for LacI


Part:BBa_K324052 - pLac/Mnt-RBS-GAL4-ECFP-Term

This part is composed of the pLac/Mnt hybrid promotor with GAL4 and ECFP following. Expression occurs in the absence of Mnt + presence of LacI and IPTG.


Part:BBa_K324053 - Constitutive-RBS-LovTAP-Term

This part is designed to constitutively express the LovTAP protein (the light responsive protein BBa_K191006)

Design Notes

This part is modified from BBa_K191003, which contained pLac. As we desired constant production of LovTAP, this promotor was replaced. See here for more information regarding the role this part plays in our circuit.

Source

BBa_K191006 and BBa_K191003

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