Assembly Workplan: The Initiator

Because the initiator is one of the smaller constructs, we have decided to use this as an example of the utmost basic workplan.

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.

• Starting Up
• RBS to c0051
• 'RBS & c0051' to stop
• 'RBS & c0051 & stop' to R0082

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! :)