The objective of this tutorial is to teach you how to turn a coding sequence into a Golden Braid modular cloning part for your parts library. Golden Braid involves a specific cloning grammar which allows for the construction of multiple-part circuits (world record was 52 parts at once!) in a LEGO-like fashion.
The above figure may look intimidating, but take a moment to look at the columns denoted by four basepairs.
These are the overhangs Golden braid relies on to assemble its parts to make domesticated parts and Transcription Units (TU’s). A basic TU involves a promoter, a coding sequence, and a terminator. Seen below is the section of the diagram that indicates the span of each part. The ends of those blocks align with one of the overhangs listed above.
A Golden Braid part will have two overhangs, a 5’ and a 3’ (pronounced five prime and three prime). Here we see that a part consisting of a Promoter + 5’ Untranslated Region + ATG start codon has the GGAG 5’ overhang and the AATG 3’ overhang.
A coding sequence (CDS) part has the AATG 5’ overhang and the GCTT 3’ overhang.
Finally, the terminator part has the GCTT 5’ overhang and the CGCT 3’ overhang.
If you have not noticed already, each subsequent part’s 3’ overhang is the 5’ overhang on the next part in the circuit. In Golden Braid, only parts with matching overhangs will ligate. If you have mutiple parts in a single tube, they will only assemble in the order designated by their corresponding overhangs such that part orientation is conserved. Let’s go back to the full length TU in the diagram to explain the last overhang pair.
So far the 3’ end of a promoter part ligates to the 5’ end of the CDS part. The 3’ end of the CDS part ligates to the 5’ end of the Terminator part. But what happens to the overhangs at the ends of a complete Transcription Unit? The GGAG and CGCT overhangs cannot ligate together so what do they stick to?
The Entry Vector!
Golden Braid has four entry vectors (A1, A2, O1, and O2) that can be cloned into if you have enough parts to make a full Transcription Unit. When you digest any of the vectors with the enzyme corresponding to that plasmid, you open up the plasmid to make a linear fragment with a GGAG 5’ and CGCT 3’ overhangs. These line up perfectly with the overhangs of a complete TU, so if you liagate all the parts of the TU and the entry vector together in a single reaction, the parts and plasmid backbone come together to form a closed loop - a complete circular plasmid.
As we can see from what we have learned so far, the multipart assembly heavily relies on your parts being formatted with the correct overhangs and correct restriction sites to enable the cutting of your part to make said overhangs “sticky” (phosphorylated). To ensure we have properly formatted parts we have to DOMESTICATE that sequence. In this tutorial we will learn how to domesticate a coding sequence so that we can add it to our parts library. Let’s get started!
Identify the coding sequence you wish to domesticate. Be sure you have a way of physically obtaining the DNA in question, otherwise you will need to synthesize the sequence from scratch. In this tutorial we will be lifting a gene from the E. coli K-12 genome, speE (spermidine synthase).
Download an annotated copy of the genome or genetic region of your organism of interest, where your gene of interest is somewhere in that file. I try to check Genbank first but feel free to use any database.
