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Gibson Assembly

This protocol demonstrates cloning of a Vibrio fischeri gene into the IPTG-inducible vector pEVS143.

Construct design

Here we use the NEBuilder to assemble the desired construct in silico.

  • Go to NEBuilder online tool
  • Click Set Preferences tab
  • Click Change Prefs
  • Set Min Primer Length to 22
  • Click Done
  • Click Build Construct tab
  • Click Add Fragment
  • Insert FASTA sequence of pEVS143 and name sequence
  • Select Make this the vector backbone
  • Click Continue
  • Select linearize by PCR
  • Select define insert site by Sequence Position
  • Insert positions of upstream and downstream flanking bases
  • Click Done
  • Add another fragment
  • Insert FASTA sequence of gene and name sequence
  • Click Continue
  • Select produce DNA by PCR
  • Click Done
  • Repeat for 2-3 fragments if needed
  • Click Get Assembly and download pdf file

  1. Order primers generated by in silico assembly to amplify linearized vector and insert(s)

  2. Resuspend lyophilized primers to a concentration of 50uM in buffer TE

Amplification and Gibson Assembly

  1. Prepare PCR reactions using polymerase Q5 and conditions laid out by Excel spreadsheet 'PCR Worksheet’

  2. Run PCR reactions in thermocycler as per cycling conditions laid out in spreadsheet

  3. To confirm PCR amplification run the samples on a 1% agarose gel

  • Mix 2ul reaction with 3 ul nuclease free water and 1 ul 6x loading dye
  • Image gel on AlphaImager HP

  1. If PCR was successful, purify reactions using QIAgen PCR Purification Kit

  2. Treat the PCR amplified vector with Dpn1 as follows:

  • In a 10ul total reaction volume use 1ul 10xCutSmart buffer, 1ul Dpn1 and 8ul PCR purified vector
  • Incubate at 37C for 1 hr
  • Heat inactivate Dpn1 at 80C for 20 min

  1. Quantify all DNA on Nanodrop

  2. Set up Gibson assembly reactions as follows

  • Thaw an aliquot of 10 ul Gibson Assembly Master Mix(2X, NEB#E2611L)
  • Add X ul of PCR fragments and linearized vector such that vector is at 50-100 ng and insert(s) is at 3-fold molar excess
  • Add 10-X ul of nuclease free water to a total reaction volume of 20 ul
  • Incubate in thermocycler at 50C for 20 minutes
  • Store samples on ice or -20C for subsequent transformation
  • Also perform the above on an insert-free/vector-only control reaction

Transformation

Transform the above into electrocompetent DH5a-lambda-pir cells as follows

  • Bring SOC media from 4C to room temperature
  • Chill electroporation cuvette on ice
  • Obtain 50ul aliquot of electrocompetent cells from -80C freezer and thaw on ice for 5 mins
  • If using DNA in a salty buffer or a ligation reaction, remember to dialyze sample before adding to cells (samples with salt will cause arcing during electroporation and kill the cells). Samples were dialyzed using Millipore Nitrocellulose Membrane Filters (#VSWP01300). ddH2O was taken in a clean, empty petri dish and one filter disc was allowed to float on the surface of the water. 10 ul of sample is pipetted onto filter disc and allowed to stand for 10 mins. The sample is then pipetted off the disc and is now ready for transformation. If using the Gibson Assembly mix, dilute 5ul in 10ul DNA-water.
  • Gently pipet 1-5 ul of DNA into cells (I usually use 1 ul of purified plasmid or 3 ul of ligation reaction)
  • Gently transfer cells to electroporation cuvette - into the narrow part at the bottom of the cuvette - being careful not to introduce air bubbles as that will cause arcing during electroporation
  • Using the Seifert lab electroporator:
  • Turn on the machine
  • Set capacitance to 25, resistance to 200 and voltage to 1.8kV (for a 1mm cuvette)
  • Remove cap from cuvette and wipe down the condensation from the sides using a kimwipe
  • Pull out sliding arm and place cuvette in holder, notch side aligned to the slit and metals making electrical contact on the sides
  • Slide holder in until the cuvette clicks into place
  • Press two red buttons simultaneously till the pulse sound is heard for about 3 seconds
  • Pull out sliding arm and immediately add 900ul SOC media and transfer cells into a sterile 1.7 ml microcentrifuge tube
  • Dispose of cuvette as glass waste and turn off the machine
  • Recover cells by incubating in 37C shaker for 1 hour
  • For purified plasmid, plate 100ul of cells on agar containing the appropriate selection. For ligation reaction, spin down cells for 5 min at 5000 rpm and remove excess media, resuspending in 100 ul media and plating on agar containing the appropriate selection.
  • Spread cells using glass beads and incubate plate at 37C overnight or until colonies start to appear
  • Include an intact vector control

  1. Check plates for clones the following day. Ideally the gibson reactions will have given several colonies while the vector-only control will have none.

  2. Screen colonies for insert by colony PCR, patching colonies onto an agar plate with appropriate selection prior to using as PCR template

  3. Colonies that give bands corresponding to the correct molecular weight will be grown up and the plasmid extracted by QIAgen Miniprep kit and submitted for sequencing using the appropriate sequencing primers

  4. Sequences will be analyzed using Seqbuilder and clones carrying the correct sequences will be stored as glycerol stocks in the -80C freezer