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gibson_assembly.md

How to clone GeneStrings into pET29b(+) using Gibson assembly

Backbone preparation

Perform a double digest of XhoI and NdeI on pET29b+ and purify the resultant 4.5 kb fragment (the backbone) using a gel purification kit. Dilute the prepared backbone to 40 ng/µL for downstream applications.

Preparing synthetic genes

Order your codon-optimized synthetic gene sequence from your vendor of choice (we use Life Technologies in the Siegel group) with the following sequences tacked on to the ends (these are specific to pET29b(+))

5' GAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATG

3' CTCGAGCACCACCACCACCACCACTGA

The 5' adapter above contains the initial methionine in the protein sequence to ensure the fragment gets inserted in frame with the pET29 backbone, so remove the initial methionine from your amino acid sequences when ordering.

How to design a synthetic gene for cloning into pET29b(+)

In general, the ordered sequence is

5' adapter + coding sequence + 3' adapter

Where coding sequence stands for the nucleotide sequence of your gene. To design a nucleotide sequence, use the Life Technologies GenArt tool (or any software you like) to design a codon-optimized nucleotide sequence from the amino acid sequence of your desired gene. Tell the software to avoid restriction sites for XhoI and NdeI when designing the nucleotide sequence. (There is also a large order assistant for batch entry of genes on Life's website.)

For example, using pET29b(+), the 5' Gibson adapter is

GAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATG

and the 3' adapter is

CTCGAGCACCACCACCACCACCACTGA

(If you need the resultant sequence to be an exact multiple of 3, when ordering IDT Gene Blocks, for example, you can remove a base or two from the beginning of the 5' Gibson adapter or the end of the 3' adapter.)

Example using pET29(+) and FabI from Escherichia coli

I used Life Technologies' codon optimization tool to optimize this original amino acid sequence (Uniprot P0AEK4) coding for Enoyl-[acyl-carrier-protein] reductase [NADH] FabI from Escherichia coli

>P0AEK4
MGFLSGKRIL VTGVASKLSI AYGIAQAMHR EGAELAFTYQ NDKLKGRVEE FAAQLGSDIV
LQCDVAEDAS IDTMFAELGK VWPKFDGFVH SIGFAPGDQL DGDYVNAVTR EGFKIAHDIS
SYSFVAMAKA CRSMLNPGSA LLTLSYLGAE RAIPNYNVMG LAKASLEANV RYMANAMGPE
GVRVNAISAG PIRTLAASGI KDFRKMLAHC EAVTPIRRTV TIEDVGNSAA FLCSDLSAGI
SGEVVHVDGG FSIAAMNELE LK

(note the N-terminal methionine), avoiding the restriction sites EcoRI, NdeI, PstI, SpeI, and XhoI, and obtained the following nucleotide sequence:

>P0AEK4 (codon-optimized for E. coli)
ATGGGTTTTCTGAGCGGTAAACGTATTCTGGTTACCGGTGTTGCAAGCAAACTGAGCATTGCCT
ATGGTATTGCACAGGCAATGCATCGTGAAGGTGCAGAACTGGCATTTACCTATCAGAACGATAA
ACTGAAAGGTCGCGTTGAAGAATTTGCAGCACAGCTGGGTAGCGATATTGTTCTGCAATGTGAT
GTTGCAGAAGATGCAAGCATTGATACCATGTTTGCCGAACTGGGTAAAGTTTGGCCGAAATTTG
ATGGTTTTGTGCATAGCATTGGTTTTGCACCGGGTGATCAGCTGGATGGTGATTATGTTAATGC
AGTTACCCGTGAAGGCTTTAAAATCGCACATGATATTAGCAGCTATTCCTTTGTTGCAATGGCA
AAAGCATGTCGTAGCATGCTGAATCCGGGTAGCGCACTGCTGACCCTGAGCTATCTGGGTGCCG
AACGTGCAATTCCGAACTATAATGTTATGGGTCTGGCCAAAGCAAGCCTGGAAGCAAATGTTCG
TTATATGGCAAATGCAATGGGTCCGGAAGGTGTTCGTGTTAATGCCATTAGCGCAGGTCCGATT
CGTACCCTGGCAGCAAGCGGTATTAAAGATTTTCGTAAAATGCTGGCACATTGCGAAGCCGTTA
CCCCGATTCGTCGTACCGTTACCATTGAAGATGTTGGTAATAGCGCAGCATTTCTGTGTAGCGA
TCTGAGCGCAGGTATTAGCGGTGAAGTTGTTCATGTTGATGGTGGTTTTAGCATTGCAGCAATG
AATGAACTGGAACTGAAA

Note the first codon, ATG, coding for methionine, is duplicated on the 5' Gibson adapter sequence above, so I will remove it from the nucleotide sequence in the next step.

Next, add the 5' Gibson adapter to the beginning of the sequence and the 3' adapter to the end, like so (plus signs shown for clarity!)

GAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATG 

+

   GGTTTTCTGAGCGGTAAACGTATTCTGGTTACCGGTGTTGCAAGCAAACTGAGCATTGCCT
ATGGTATTGCACAGGCAATGCATCGTGAAGGTGCAGAACTGGCATTTACCTATCAGAACGATAA
ACTGAAAGGTCGCGTTGAAGAATTTGCAGCACAGCTGGGTAGCGATATTGTTCTGCAATGTGAT
GTTGCAGAAGATGCAAGCATTGATACCATGTTTGCCGAACTGGGTAAAGTTTGGCCGAAATTTG
ATGGTTTTGTGCATAGCATTGGTTTTGCACCGGGTGATCAGCTGGATGGTGATTATGTTAATGC
AGTTACCCGTGAAGGCTTTAAAATCGCACATGATATTAGCAGCTATTCCTTTGTTGCAATGGCA
AAAGCATGTCGTAGCATGCTGAATCCGGGTAGCGCACTGCTGACCCTGAGCTATCTGGGTGCCG
AACGTGCAATTCCGAACTATAATGTTATGGGTCTGGCCAAAGCAAGCCTGGAAGCAAATGTTCG
TTATATGGCAAATGCAATGGGTCCGGAAGGTGTTCGTGTTAATGCCATTAGCGCAGGTCCGATT
CGTACCCTGGCAGCAAGCGGTATTAAAGATTTTCGTAAAATGCTGGCACATTGCGAAGCCGTTA
CCCCGATTCGTCGTACCGTTACCATTGAAGATGTTGGTAATAGCGCAGCATTTCTGTGTAGCGA
TCTGAGCGCAGGTATTAGCGGTGAAGTTGTTCATGTTGATGGTGGTTTTAGCATTGCAGCAATG
AATGAACTGGAACTGAAA

+ 

CTCGAGCACCACCACCACCACCACTGA

and the result is your sequence to order:

GAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGGGTTTTCTGAGCGGTAAACGTAT
TCTGGTTACCGGTGTTGCAAGCAAACTGAGCATTGCCTATGGTATTGCACAGGCAATGCATCGT
GAAGGTGCAGAACTGGCATTTACCTATCAGAACGATAAACTGAAAGGTCGCGTTGAAGAATTTG
CAGCACAGCTGGGTAGCGATATTGTTCTGCAATGTGATGTTGCAGAAGATGCAAGCATTGATAC
CATGTTTGCCGAACTGGGTAAAGTTTGGCCGAAATTTGATGGTTTTGTGCATAGCATTGGTTTT
GCACCGGGTGATCAGCTGGATGGTGATTATGTTAATGCAGTTACCCGTGAAGGCTTTAAAATCG
CACATGATATTAGCAGCTATTCCTTTGTTGCAATGGCAAAAGCATGTCGTAGCATGCTGAATCC
GGGTAGCGCACTGCTGACCCTGAGCTATCTGGGTGCCGAACGTGCAATTCCGAACTATAATGTT
ATGGGTCTGGCCAAAGCAAGCCTGGAAGCAAATGTTCGTTATATGGCAAATGCAATGGGTCCGG
AAGGTGTTCGTGTTAATGCCATTAGCGCAGGTCCGATTCGTACCCTGGCAGCAAGCGGTATTAA
AGATTTTCGTAAAATGCTGGCACATTGCGAAGCCGTTACCCCGATTCGTCGTACCGTTACCATT
GAAGATGTTGGTAATAGCGCAGCATTTCTGTGTAGCGATCTGAGCGCAGGTATTAGCGGTGAAG
TTGTTCATGTTGATGGTGGTTTTAGCATTGCAGCAATGAATGAACTGGAACTGAAACTCGAGCA
CCACCACCACCACCACTGA

Once you receive the gene string

Resuspend dried-down DNA Strings to 20 ng/µL with nuclease-free water. (To calculate, divide the weight of dried-down DNA in nanograms by 20 to obtain the number of micoliters to resuspend in. For example, you recieve a gene that is 1150 ng dried down. Resuspend in 1150/20=57.2 µL to get a final concentration of 20 ng/µL.

It's recommended to do 5 µL Gibsons with 20 ng of backbone and 30 ng of insert, provided that the insert (~1500 bp) is roughly 2-3 fold smaller than the backbone (~4500 bp).

Nominally, we want a 1 to 1 molar ratio of pieces for Gibson, but in the case of inserting a small fragment (~1500 bp) into a larger fragment (~4500 bp), we want 2-3 fold excess of insert for high efficiency. Since the fragment's ~1500 bp is approximately 2-3 fold smaller than the backbone, it happens that a equal weight of insert and backbone is ideal for the kind of Gibsons we typically in our lab. (Of course, if your insert is not near ~1500 bp, you will need to calculate the weight that provides a 2-3 fold molar excess.)

Gibson master mix

Purchase Gibson master mix from NEB (catalog number E2611S).

Assembly procedure

Create PCR program:

  • 4 deg C for ever
  • 50 deg C for 60 min
  • 4 deg C for ever

Lid temp: 50 C, volume: 5 µL.

Make a master mix of the backbone and Gibson mix.

  • Keep in 4 C thermal cycler/on ice at all times!
  • Make enough for n+1 assemblies
  • Dilute the backbone to 13.3 ng/µL beforehand with nuclease-free water.

In PCR strip tube, combine

  • Gibson mix: 2.5 µL * n_reactions
  • Linearized backbone (concentration=13.3 ng/µL): 1.5 µL * n_reactions

to a final volume of 4 µL * n_reactions.

  1. Aliqot 4 µL of this master mix to PCR strip at 4 C in thermal cycler
  2. Aliout 1 µL of your resuspended GeneStrings
  3. Seal with caps
  4. Mix by flicking and spinning down in baby centrifuges for 1 second
  5. Put PCR strips back in thermal cycler
  6. Tap "Skip step" to move to the 1 hour 50 C incubation
  7. After 1 hour, dialyze

Dialysis of Gibson mixture

  1. Float a 0.022 micron filter in a Petri dish filled with MilliQ water
  2. Pipet the 5 uL reaction on to the floating paper filter
  3. Incubate at room temperature for 1-2 hours
  4. Pipet the drop into a fresh PCR tube
  5. Electroporate 1 uL of the mix into DH5a and freeze the rest
Raw
resuspend_gibson_meez.md

Resuspend and Gibson assembly of dried-down DNAStrings

Mise en place

  • dried down tubes
  • nuclease free water
  • small centrifuge
  • P200
  • medium tips

Procedure

  • For each tube, resuspend DNA in 100 µL nuclease-free water (alternatively, resuspend to 20 ng/µL with nuclease free water).

  • Make a master backbone mix. You will need 4(n+1) µL of 1X Gibson assembly mix, 20 ng/µL linearized backbone. Aliquot 4 µL into PCR strips, add in 1 µL of resuspended DNA. Flick and spin down. Run PCR program 50 ˚C for 1:00:00, 4 ˚C for ∞.

  • Dialyze the Gibson assemblies for 1-2 hours at room temperature. Recover 5-6 µL from the dialysis membrane.

  • Transform 1 µL into E. coli DH10B using electroporation, recover 1-2 hours, and plate on kanamycin selection plates.

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