Assembly Workplans

Creating Each Individual Composite Part

**Click on the individual pieces below to view how we ligated them together.**

Miscellaneous Parts

Genetic Circuit Pieces

Next Mission: Placing them into only two plasmids

Final Mission: Create an Even Lawn of E. Coli

• We are using this top agar plating technique
• Next is to test the what the optimal OD, arabinose concentration, and amount of light it takes to a) create an even lawn of E. Coli and b) see contrast between the "on" and "off" cells

Basic Assembly Protocols

For more insight about the process behind the ligations, here is a sample ligation procedure. Because the initiator is one of the smaller constructs, we have decided to use it as an example to illustrate the protocols used in lab.

Starting Up

• Hydrated parts: BBa_B0034, BBa_C0051, BBa_B0015, BBa_R0082.
• Transformed these parts into DH5α competent cells.

RBS to BBa_C0051

• Cultured cells that have parts: BBa_B0034 and BBa_C0051
• Miniprepped BBa_B0034 and BBa_C0051

Digest with SpeI & Pst Run on gel Gel extract

Digest with Xba & Pst Separate fragments through gel electrophoresis Gel extract

• Ligate the RBS and BBa_C0051 fragments together and transform on carb plates.
• Depending on the outcome of visible colonies on the experimental and control plates, a PCR screening may be needed.

• It is most practical to cut RBS as a vector because RBS is less than 100 bp long as an insert, making it hard to see and extract on the gel.
• The sticky ends from the XbaI and SpeI, when ligated together, will form an 8 bp scar. Thus, XbaI and SpeI will no longer be able to cut here.

• Cultured cells that have parts: 'RBS & BBa_C0051', BBa_B0015
• Miniprepped 'RBS & BBa_C0051' and BBa_B0015

Digest with EcoRI & XbaI Run on gel Gel extract

Digest with EcoRI & SpeI Separate fragments through gel electrophoresis Gel extract

• Ligate the fragments together and transform on kan plates.
• Depending on the outcome of visible colonies on the experimental and control plates, a PCR screening may be needed.

Assembly notes

• It is most practical to cut BBa_B0015 as the vector because it is only 130 bp long. It would be hard to see on a gel.

• Cultured cells that have parts: 'RBS & BBa_C0051 & stop', BBa_R0082
• Miniprepped 'RBS & BBa_C0051& stop' and BBa_R0082

Digest with SpeI & PstI Run on gel Gel extract

Digest with XbaI & PstI Separate fragments through gel electrophoresis Gel extract

• Ligate the fragments together and transform on carb plates.
• Depending on the outcome of visible colonies on the experimental and control plates, a PCR screening may be needed.

• The promoter had to be cut as a vector because it is only 108 bp by itself.

• Miscellaneous Parts
• Main Genetic Circuit
• Sample Ligation Procedure

We would also like to thank and acknowledge:
Our Advisors
Marc Facciotti
Ilias Tagkopoulos
Technical Guidance
David Larsen
Andrew Yao
Visiting iGEMer
Jia Li of Zhejiang University (TEAM ZJU-China)
cI Promoter Screen
Drew Endy - Stanford
Thomas Schneider - NIH
Want to sponsor us? Send an email to mtfacciotti@ucdavis.edu to discuss various ways you can help! :)