(r7) Barrick Lab :: ProtocolsGoldenGateMakingANewPart

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---+!! Golden Gate Assembly: Creating a New Part

This protocol describes how to clone a new Golden Gate part amplified from a template DNA (like a plasmid or a genome) or synthesized as a piece of double-stranded DNA (like an IDT gBlock) into the YTK001 entry vector via a BsmBI assembly reaction.
 
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---++ Supplies

   * dsDNA piece encoding the part with restriction sites appended (see below)
   * Phusion® High-Fidelity DNA Polymerase (NEB: M0530)
   * 10&times; T4 DNA ligase buffer (NEB: M0202)
   * T7 DNA ligase (NEB: M0318)
   * Restriction endonuclease BsmBI (NEB: R0580)
   * Competent cells
   * SOC and liquid media for recovery after transformation
   * LB+Cam plates for recovering transformants with assembled plasmid

---++ Step 1: Creating dsDNA encoding the part for cloning

Two variations on preparing a dsDNA fragment with the proper restriction sites and Golden Gate overhangs are provided in the next sections. The end result is the same: a piece of DNA with the proper flanking regions for BsmBI cloning into the entry vector, while maintaining the BsaI sites used in first stage assembly – with proper overhangs for the type of part that you are designing.

---+++ Method 1: Synthesizing dsDNA containing the required flanking regions

When ordering a double-stranded piece of DNA to be synthesized, you can just append the sites needed for cloning in your order. Use this protocol when ordering a gBlock from IDT, for example.

<div style="font-family:courier; line-height: 100%;">5'-GCAT%GREEN%CGTCT%ENDCOLOR%CATCG%BLUE%GTCTC%ENDCOLOR%A%RED%XXXX%ENDCOLOR% YOUR PART %PURPLE%YYYY%ENDCOLOR%T%BLUE%GAGAC%ENDCOLOR%CTG%GREEN%AGACG%ENDCOLOR%GCAT-3'%BR%
3'-CGTA%GREEN%GCAGA%ENDCOLOR%GTAGC%BLUE%CAGAG%ENDCOLOR%T%RED%xxxx%ENDCOLOR% your part %PURPLE%yyyy%ENDCOLOR%A%BLUE%CTCTG%ENDCOLOR%GAC%GREEN%TCTGC%ENDCOLOR%CGTA-5'</div>

Replace <font style="font-family:courier; line-height: 100%;">%RED%XXXX%ENDCOLOR%</font> with the prefix-F sequence for the part type you are designing (*see the table and notes below*). Similarly, replace
<font style="font-family:courier; line-height: 100%;">%PURPLE%YYYY%ENDCOLOR%</font> with the suffix-F sequence for the part.

In general, the amount of DNA synthesized is sufficient for cloning into the entry vector without further PCR amplification of a gBlock.

---+++ Method 2: Amplifying a sequence with primers that add the required flanking regions

You need to order two primers that append sequences to the ends of the DNA sequence that you are amplifying. If your template looks like the following, then you should design the two primers like the examples.

*Template*
<div style="font-family:courier; line-height: 100%;">5'-UUUUUUUUUUUUUUUUUUUU YOUR PART DDDDDDDDDDDDDDDDDDD-3'
%BR%3'-uuuuuuuuuuuuuuuuuuuu your part ddddddddddddddddddd-5'</div>

*Primer 1 (forward)*
<div style="font-family:courier; line-height: 100%;">5'-GCAT%GREEN%CGTCT%ENDCOLOR%CATCG%BLUE%GTCTC%ENDCOLOR%A%RED%XXXX%ENDCOLOR%UUUUUUUUUUUUUUUUUUUU-3'</div>

*Primer 2 (reverse)*
<div style="font-family:courier; line-height: 100%;">5'-ATGC%GREEN%CGTCT%ENDCOLOR%CAG%BLUE%GTCTC%ENDCOLOR%A%PURPLE%yyyy%ENDCOLOR%ddddddddddddddddddd-3'</div>

*CAUTION* In Primer 2, the priming site (dddd...) must be the reverse-complement of your part and you must use the *suffix-R* sequence for the Golden Gate part overlap because this is on the other strand.

The overlap with the template can vary from the 20 base pairs that are shown according to the normal rules for designing good PCR primers. If calculating melting temperatures, be sure to only include the overlap region in your calculations, not the stuff that is being added to the ends!

---++++ Example PCR Reaction

   * PCR Insert
   * Use standard 25ul Phusion (or other high fidelity polymerase) protocol
   * PCR (25ul reaction)
   * 5 ul 5x Buffer
   * 1.5 ul dntps
   * 1.25 ul primer (A+C)
   * 1.25 ul primer (B+D)
   * x ul template plasmid (<250ng)

ddH20 to 24.5 µl

then add 0.25 µl Phusion

Set elongation time according to size of insert. 
Purify PCR products

*CAUTION* The plasmid you are constructing in the entry vector (pYTK001) is camR, so if your template plasmid also encodes chloramphenicol resistance, you should DpnI digest after your PCR reaction, and probably also gel purify the fragment, as any residual plasmid may be transformed and lead to false-positive colonies.

---+++ Part Overlap Quick Reference Table

These part overhangs are used by the YTK (yeast toolkit) and the BTK (bee toolkit). More information on the design of parts for Golden Gate assembly using these standards can be found in the reference to the YTK (especially in the supplement). Be aware that some definitions vary between the two toolkits.

| Type | Description | Prefix (F) | Prefix (R) | Suffix (F) | Suffix (R) | Notes | 
| 2 | promoter + RBS | AACG | cgtt | TATG | cata |  |
| 3 | (BTK) gene | TATG | cata | ATCC | ggat | The start codon (ATG) is within the prefix. After that, begin your protein coding sequence in-frame with the second codon. BTK: *Include the stop codon for your gene!*. |
| 3 | (YTK) gene | TATG | cata | %RED%GG%ENDCOLOR%ATCC | ggat%RED%cc%ENDCOLOR% | The start codon (ATG) is within the prefix. After that, begin your protein coding sequence in-frame with the second codon. YTK: Omit the stop codon. It  is typically placed in the next part. For that stop codon to be in-frame, you must end this part with the two bases GG so that plus the suffix encode a Gly-Ser linker/extension. |
| 4 | (BTK) terminator | ATCC | ggat | GCTG | cagc |  |
| 4 | (YTK) stop codon + terminator | ATCC%RED%TAA%ENDCOLOR% | %RED%tta%ENDCOLOR%ggat | GCTG | cagc | The first three bases within the part should be a stop codon (TAA). |

   * All sequences in the table are written 5' to 3'. 
   * YTK: yeast toolkit; BTK: bee toolkit.
   * Standard bases that are added before or after the actual Golden Gate overhang in prefix / suffix to maintain part function are shown in red.

---++ Step 2: Golden Gate Assembly Reaction

Now, you perform a Golden Gate Assembly reaction with BsmBI to ligate your part into the entry vector (pYTK001).

   1 Mix 17.7ng pYTK-001 plasmid  and 20 fmol=[(0.02*(660)*(1/(10^6))*insert length)*1000]ng insert of your DNA fragments together.
   1 Add that 2&#956;L of 10&times; T4 DNA ligase buffer, 1 &#956;L of BsmBI, 1 &#956;L of T7 DNA ligase , and add water up to 20 &#956;L, mix well by pipetting.
   1 Incubate the reaction at (42°C for 5 min and then 16°C for 5 min) *30 , followed by 10 min at 55°C, 10min at 80 °C .
   1 Use 2 µl assembly reaction for electroporation.
   1 After recovery, plate on LB+Cam.

---++ *References*

   1 Lee ME, DeLoache WC, Cervantes B, Dueber JE. (2015) A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. _ACS Synth. Biol._ *4*:975–986. [[http://pubs.acs.org/doi/abs/10.1021/sb500366v][Link]]

---++ *Contributors*

   * Peng Geng
   * Jeffrey Barrick
Barrick Lab > ProtocolList > ProtocolsGoldenGateMakingANewPart
Contributors to this topic
KateElston, JeffreyBarrick, DennisMishler, PengGeng, GabrielSuarez, SeanLeonard
Topic revision: r7 - 2016-05-16 - 03:18:29 - Main.JeffreyBarrick