(r14) Barrick Lab :: ProtocolsBTKAssembleSingleTUPlasmid

[[GoldenGateAssemblyProtocolsMainPage][Back to Golden Gate Protocols]]

---+ First-Stage Plasmid (Transcriptional Unit) Assembly

Once you have all your desired part plasmids built you can assemble them into Transcriptional units (TU). Typically these plasmids will be assembled with part plasmids for promoter + RBS, coding sequence, terminator, and TU specific connectors; variations on this basic strategy are easily managed though as long as the requisite overhangs are present ([[http://barricklab.org/twiki/bin/view/Lab/ProtocolsBTKDesignANewPart Described here]]).

---+++ Assembly reaction

Total volume will be 20 &mu;L; you will need 10 fmol of each part plasmid for this assembly. Typically the pYTK095 Type 6-8 plasmid (ColE1 origin, sfGFP dropout) is used as the backbone for this assembly. If toxicity issues may be a factor, lower copy number vectors can easily be used in its place.

   * 10 fmol of each part plasmid = 650 * insert length * 10x10^-6 = *X ng*
   * 2 &mu;L of 10× T4 DNA ligase buffer (Promega)
   * 1 &mu;L of BsaI-HFv2 (**DO NOT* use BsaI or BsaI-HF)
   * 1 &mu;L of T4 DNA ligase (Promega)
   * x &mu;L water up to 20 &mu;L total.

For easy part volume calculation: [[%ATTACHURL%/BroadHostRange_Reaction_Calculator.xlsx][BroadHostRange_Reaction_Calculator.xlsx]]

*BsaI and BsaI-HF don't have high fidelity and/or activity under the Golden Gate buffer/temperature conditions.

** Promega T4 Ligase buffer has been shown to offer the highest activity for BsaI enzyme. Other T4 ligase enzymes will work, but Promega enzyme with Promega buffer is highest efficiency.
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Mix samples well by pipetting, then run the reaction on the thermocycler under the following conditions:

|  *Step*  |  *Temperature*  |  *Time*  |
|  1  |  37°C  |  1.5 min  |
|  2  |  16°C  |  3 min  |
| Cycles 1-2: | Repeat 25x | |
|  3  |  37°C  |  5 min  |
|  4  |  80°C  |  10 min |

 Transform 2 &mu;L of the assembly reaction and plate on an appropriate antibiotic (Amp/Carb if using pYTK095).

Tips from New England Biolabs on ways to change the reaction conditions for difficult assemblies involving many parts:
   * Different reaction times based on the number of inserts ([[https://www.neb.com/protocols/2015/03/04/golden-gate-assembly-protocol-for-using-neb-golden-gate-assembly-mix-e1600][link]])
   * Better fidelity for assembling many inserts using a constant reaction temperature: ([[https://www.biorxiv.org/content/10.1101/2020.12.22.424019v1][link]])

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Attachments

Topic attachments
I	Attachment	History	Action	Size	Date	Who	Comment
xlsx	BroadHostRange_Reaction_Calculator.xlsx	r1	manage	17.0 K	2021-07-09 - 20:56	KateElston

Barrick Lab > BroadHostRangeToolkit > ProtocolsBTKAssembleSingleTUPlasmid

Contributors to this topic

KateElston, SeanLeonard, PatrickLariviere, VictorLi, JeffreyBarrick, DennisMishler

Topic revision: r14 - 2021-07-09 - 20:58:01 - Main.KateElston