Registry/Measurement kit

The Measurement Kit consists of Promoter Testers, RBS Testers, and an Inverter/Terminator Tester. All Devices are on the 3K3 plasmid. Also, they are being built in both GFP and RFP versions. (GFP versions shown below)

Promoter Tester

E0240 Version 1 Design

The Promoter Tester using GFP is based on <BBpart> E0240</BBpart> and using RFP is based on <BBpart> I2057</BBpart>. Both come in two versions.

• Version 1 (E/S)
  • ___E X CCDB S___RBS GFP T
  • Cut promoter E/S and insert
  • Can take part back out if necessary

• Version 2
  • ___E CCDB X___E0240
  • Cut Promoter E/S to insert
  • Forms mixed site so can't remove, real conditions

• GFP Promoter Tester Version 1 E/S <BBpart> I20244</BBpart>
• GFP Promoter Tester Version 2 E/X <BBpart> I20245</BBpart>
• RFP Promoter Tester Version 1 E/S <BBpart> I20246</BBpart>
• RFP Promoter Tester Version 2 E/X <BBpart> I20247</BBpart>

RBS Tester

I2055 Design

The RBS Tester using GFP is based on <BBpart> I2055</BBpart> and using RFP is based on <BBpart> I2056</BBpart>. Both come in two versions.

• Versions 1 (E/X)
  • __R0040___E CCDB X____GFP T
  • To insert RBS cut E/S, forms a mixed Spe/Xba site
  • Has too big spacing (full BB prefix) b/t promoter and RBS
  • Easier for testers to use

• Version 2 (S/X)
  • __R0040___S CCDB X____GFP T
  • Will get 2 spe/xba mixed sites, can't get RBS out
  • Cut RBS X/S, ligate w/ Spe1 and Xba1 restriction enzymes to ensure proper orientation
  • Real condition spacing

• GFP RBS Tester Version 1 E/X <BBpart> I20248</BBpart>
• GFP RBS Tester Version 2 S/X <BBpart> I20249</BBpart>
• RFP RBS Tester Version 1 E/X <BBpart> I20250</BBpart>
• RFP RBS Tester Version 2 S/X <BBpart> I20251</BBpart>

Inverter/Terminator Tester

T9002 Design

• Based on <BBpart> T9002</BBpart>

• F2620________E X P1010 S P________E0040
• Contains a full BB site to allow construction within the device

Project Pages

• Current notebook
  • Registry/Measurement kit/Instructions
• Two summer notebooks on construction of the kit:Nishant, Karen
• Some early design notes (Karen's design notes)
• Construction approach (Primers used in construction)
• Glycerol box

References

1. Alper H, Fischer C, Nevoigt E, and Stephanopoulos G. Tuning genetic control through promoter engineering. Proc Natl Acad Sci U S A. 2005 Sep 6;102(36):12678-83. DOI:10.1073/pnas.0504604102 |
2. Zaslaver A, Bren A, Ronen M, Itzkovitz S, Kikoin I, Shavit S, Liebermeister W, Surette MG, and Alon U. A comprehensive library of fluorescent transcriptional reporters for Escherichia coli. Nat Methods. 2006 Aug;3(8):623-8. DOI:10.1038/nmeth895 |
3. Chen H, Bjerknes M, Kumar R, and Jay E. Determination of the optimal aligned spacing between the Shine-Dalgarno sequence and the translation initiation codon of Escherichia coli mRNAs. Nucleic Acids Res. 1994 Nov 25;22(23):4953-7. DOI:10.1093/nar/22.23.4953 |