TITLE: Rapid Single-Pot Assembly of Modular Chromatin Proteins for Epigenetic Engineering
AUTHORS: James Priode, Karmella Haynes

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Chromatin is the nucleo-protein complex that organizes genomic DNA in the nuclei of eukaryotic cells. Transcriptional regulators and enzymes are key mediators of chromatin states that affect gene expression and consequently cell phenotype. Many natural chromatin mediators contain sub-domains that can be isolated and recombined to build novel regulators and live cell probes. A wide variety of synthetic chromatin proteins can be constructed from the diverse array of known natural chromatin proteins and synthetic variants. The process of engineering chromatin mediators and probes produces new tools for cell engineering and deepens our understanding of the mechanism by which chromatin features, such as modifications of histones and DNA, contribute to the epigenetic states that govern DNA-templated processes. To support efficient exploration of the large combinatorial design space of synthetic chromatin proteins, we have developed a Golden Gate assembly method for one-step construction of recombinant protein-encoding DNA. A set of standard 2-amino acid linkers allow facile assembly of any combination of up to four protein modules, obviating the need to design different compatible overhangs to ligate different modules. Beginning with the identification of protein modules of interest, a synthetic chromatin protein can be built and expressed </span><span style = "font-style:italic;">in vitro</span><span> or in cells in under two weeks.</span></div></div>

[STEPS]
?. [Preparation of Donor DNA Fragments]
Generation of linear donor DNA via Q5 HiFi PCR amplification.
?. [Preparation of Donor DNA Fragments]
Obtain template DNA for the module(s) of interest that contains a complete open reading frame (ORF) without stop codons.Ensure that the sequence does not contain any BbsI recognition sites (5'-GAAGAC). If the sequence contains these sites, modify the sequence by creating a silent substitution mutation so that the affected codon corresponds to the same amino acid as the original sequence. This can be achieved with site directed mutagenesis of plasmid DNA that carries the ORF, or after cloning the fragment into the TOPO vector (Step 7), or by purchasing a de novo synthesized fragment with the desired sequence.
?. [Preparation of Donor DNA Fragments]
Preparation of diluted PCR primers. Prepare working solutions of forward and reverse primers at a final concentration of 10 μM.
?. [Preparation of Donor DNA Fragments]
Designing PCR primers to add BbsI sites to donor DNA. Design forward and reverse primers that include the appropriate restriction site and overhang sequences (see Table 1) followed by at least 15 bp (shown as 5’-NNN… in Table 1) that bind the template. Synthesize the primers as single-stranded oligos or purchase the primers from a commercial vendor. The template-binding region of the primer should have a Tm value of . Use the NEB Tm Calculator (set for Q5 High-Fidelity DNA Polymerase) and adjust the binding region as needed to achieve this Tm target value (https://tmcalculator.neb.com/#!/main).PrimerSequence (5'-3')PurposeA-Module fTTGAAGACCTGGAGNNNNNNNNNNNNNNNAdd left BbsI and 4 bp overhang onto the 5’ side of module A.A-Module rTTGAAGACCCCAGANNNNNNNNNNNNNNN
Add right BbsI and 4 bp overhang onto the 3’ side of module A.B-Module fTTGAAGACCTTCTGGANNNNNNNNNNNNNNN
Add left BbsI and 4 bp overhang onto the 5’ side of module B.B-Module rTTGAAGACCCATGGNNNNNNNNNNNNNNN
Add right BbsI and 4 bp overhang onto the 3’ side of module B.C-Module fTTGAAGACCCATGGNNNNNNNNNNNNNNN
Add left BbsI and 4 bp overhang onto the 5’ side of module C.C-Module rTTGAAGACCCGGGCNNNNNNNNNNNNNNN
Add right BbsI and 4 bp overhang onto the 3’ side of module C.D-Module fTTGAAGACCTGCCCCTNNNNNNNNNNNNNNN
Add left BbsI and 4 bp overhang onto the 5’ side of module D.D-Module rTTGAAGACCCTAGTNNNNNNNNNNNNNNN
Add right BbsI and 4 bp overhang onto the 3’ side of module D.
65 °C
PrimerSequence (5'-3')PurposeA-Module fTTGAAGACCTGGAGNNNNNNNNNNNNNNNAdd left BbsI and 4 bp overhang onto the 5’ side of module A.A-Module rTTGAAGACCCCAGANNNNNNNNNNNNNNN
Add right BbsI and 4 bp overhang onto the 3’ side of module A.B-Module fTTGAAGACCTTCTGGANNNNNNNNNNNNNNN
Add left BbsI and 4 bp overhang onto the 5’ side of module B.B-Module rTTGAAGACCCATGGNNNNNNNNNNNNNNN
Add right BbsI and 4 bp overhang onto the 3’ side of module B.C-Module fTTGAAGACCCATGGNNNNNNNNNNNNNNN
Add left BbsI and 4 bp overhang onto the 5’ side of module C.C-Module rTTGAAGACCCGGGCNNNNNNNNNNNNNNN
Add right BbsI and 4 bp overhang onto the 3’ side of module C.D-Module fTTGAAGACCTGCCCCTNNNNNNNNNNNNNNN
Add left BbsI and 4 bp overhang onto the 5’ side of module D.D-Module rTTGAAGACCCTAGTNNNNNNNNNNNNNNN
Add right BbsI and 4 bp overhang onto the 3’ side of module D.
?. [Preparation of Donor DNA Fragments]
Q5 HiFi PCR set-up. Set up the following reaction for each donor part as follows.ReagentVol (µl)Final Concentration*Q5 buffer (5X)5.01X*dNTPs (10 mM)0.50.2 mM10 µM primer F1.250.5 µM10 µM primer R1.250.5 µMDNA0.5*Q5 Polymerase0.25MB H2O16.25Total25
ReagentVol (µl)Final Concentration*Q5 buffer (5X)5.01X*dNTPs (10 mM)0.50.2 mM10 µM primer F1.250.5 µM10 µM primer R1.250.5 µMDNA0.5*Q5 Polymerase0.25MB H2O16.25Total25
?. [Preparation of Donor DNA Fragments]
Q5 HiFi PCR. Perform PCR with the following cycle conditions.Cycle NumberDenatureAnnealExtendHold198°C, 30 s2-2698°C, 10 s65°C, 30 s72°C, 30 s2772°C, 2 minPost-reaction4°C, ∞
Cycle NumberDenatureAnnealExtendHold198°C, 30 s2-2698°C, 10 s65°C, 30 s72°C, 30 s2772°C, 2 minPost-reaction4°C, ∞
?. [Preparation of Donor DNA Fragments]
DNA purification. Purify the linear donor DNA (PCR product) using the GenElute™ PCR Clean-Up Kit according to the manufacturer’s guidelines.
?. [Generation of Plasmid Donors (optional)]
Ligation of linear donors into plasmids through Zero Blunt™ TOPO™ PCR Cloning. The PCR-amplified linear donor module, now flanked by the module’s specific BbsI sites, is subjected to a Zero Blunt™ TOPO™ PCR Cloning reaction in a PCR tube according to the reaction table below. Reaction time is five minutes at room temperature.ReagentVolume (µl)Fresh PCR product0.5*Salt solution1.0*pCR-Blunt II-TOPO1.0MB H2O3.5Total6.0
ReagentVolume (µl)Fresh PCR product0.5*Salt solution1.0*pCR-Blunt II-TOPO1.0MB H2O3.5Total6.0
?. [Preparation of Donor DNA Fragments]
Gel electrophoresis confirmation of PCR amplified linear donor DNA. Cast a 1% (wt/vol) low EEO agarose gel in 1X TAE buffer with 1:10,000 SYBR Safe DNA Gel Stain. Load  of GeneRuler 1 kb DNA ladder in the first well and  of purified linear donor DNA with  MB H2O and  6X Tritrack DNA loading dye in the adjacent wells. Run the gel for  at 100V. Successful reactions should yield a single product equal to the size of the donor fragment plus overhangs (Table 1).If you are using linear DNA for Golden Gate assembly, proceed to “Golden Gate Assembly of Fusion Protein Plasmids. If you want to generate donor plasmids, proceed to “Generation of Plasmid Donors” (below).
3 µl
3 µl
2 µl
1 µl
?. [Generation of Plasmid Donors (optional)]
Transformation with Zero Blunt™ TOPO™ PCR Cloning Kit ligation products.
?. [Recombinant Plasmid Screening with Colony PCR]
Plate inspection. Obtain colony plates from the incubator and inspect for colony growth. Optimal growth for colony PCR will have clearly separated colonies with minimal to no surrounding satellites, allowing precise colony picking with no cross-contamination. Determine the number of colonies to be picked based on the following recommendations:Plasmid Donor (Zero Blunt™ TOPO™), 2 - 4 colonies per unique ligationGolden Gate ligations, 4 - 6 colonies per unique ligation
?. [Recombinant Plasmid Screening with Colony PCR]
Preparing reagents for colony PCR. Prepare the colony PCR mixture according to the reaction table below. It is highly recommended to make a batch reaction mix (Master Mix) for efficient, error-free set-up of multiple colony PCR reactions. Multiply the volume of each reagent by the number of colonies to be screened plus one to account for pipetting error (n + 1).PrimerSequencePurposeM13 fGTAAAACGACGGCCAGForward primer for PCR-Blunt II-TOPO (5’ EcoRI site), and GGDest1-Amp, GGDest2-Amp, and GGDest3-Amp (5’ BbsI site).M13 rCAGGAAACAGCTATGACReverse primer for PCR-Blunt II-TOPO (3’ EcoRI site), and GGDest1-Amp, GGDest2-Amp, and GGDest3-Amp (3’ BbsI site).GGDestX1-Amp FGTGATAATGGTTGCAGCTAGCForward primer for GGDestX1-Amp (5’ BbsI site).GGDestX1-Amp RGGCTTTGCTCGAGTTAGCReverse primer for GGDestX1-Amp (3’ BbsI site).GGDestX2-Amp FTTTCAGGTTGGACCGGTGForward primer for GGDestX2-Amp (5’ BbsI site).BGHRTAGAAGGCACAGTCGAGGReverse primer for GGDestX2-Amp (3’ BbsI site).U6GACTATCATATGCTTACCGTForward primer for GGDestX2-Amp (U6 promoter, at 5’ of gRNA drop-in site).ReagentVolume per Colony (µL)Master MixFinal ConcentrationForward primer 10 µM1.01.0 x (n + 1)0.4 µMReverse primer 10 µM1.01.0 x (n + 1)0.4 µMDreamTaq Green PCR Master Mix (2X)12.512.5 x (n + 1)1XMB H2O10.510.5 x (n + 1)Total25.025.0 x (n + 1)
PrimerSequencePurposeM13 fGTAAAACGACGGCCAGForward primer for PCR-Blunt II-TOPO (5’ EcoRI site), and GGDest1-Amp, GGDest2-Amp, and GGDest3-Amp (5’ BbsI site).M13 rCAGGAAACAGCTATGACReverse primer for PCR-Blunt II-TOPO (3’ EcoRI site), and GGDest1-Amp, GGDest2-Amp, and GGDest3-Amp (3’ BbsI site).GGDestX1-Amp FGTGATAATGGTTGCAGCTAGCForward primer for GGDestX1-Amp (5’ BbsI site).GGDestX1-Amp RGGCTTTGCTCGAGTTAGCReverse primer for GGDestX1-Amp (3’ BbsI site).GGDestX2-Amp FTTTCAGGTTGGACCGGTGForward primer for GGDestX2-Amp (5’ BbsI site).BGHRTAGAAGGCACAGTCGAGGReverse primer for GGDestX2-Amp (3’ BbsI site).U6GACTATCATATGCTTACCGTForward primer for GGDestX2-Amp (U6 promoter, at 5’ of gRNA drop-in site).
ReagentVolume per Colony (µL)Master MixFinal ConcentrationForward primer 10 µM1.01.0 x (n + 1)0.4 µMReverse primer 10 µM1.01.0 x (n + 1)0.4 µMDreamTaq Green PCR Master Mix (2X)12.512.5 x (n + 1)1XMB H2O10.510.5 x (n + 1)Total25.025.0 x (n + 1)
?. [Recombinant Plasmid Screening with Colony PCR]
Colony PCR.
?. [Recombinant Plasmid Screening with Colony PCR]
Agarose Gel Electrophoresis. Cast a 1% (wt/vol) low EEO agarose gel in 1X TAE buffer with 1:10,000 SYBR Safe DNA Gel Stain. Load   of GeneRuler 1 kb DNA ladder in the first well and  of each colony PCR product in the remaining wells. Run the gel at 110 V for .
3 µl
15 µl
?. [Recombinant Plasmid Screening with Colony PCR]
Gel Imaging and confirmation of colony PCR amplicons.
?. [Recombinant Plasmid Preparation and Verification with Restriction Digests]
Liquid cultures of colony PCR-confirmed plasmid donor colonies. Obtain the colony streak plate and identify the streaks confirmed with colony PCR. Prepare 14 mL liquid culture tubes for the colonies to be grown by pipetting  to  of growth medium (supplemented with the appropriate antibiotic) into each tube. Pick bacteria from the streaks with sterile pipette tips and place the tips into their respective tubes. Place tubes on a shaking rack in a  incubator for  (NEB DH5ɑ-Turbo) to  (other strains, e.g. BL21).
3 mL
5 mL
37 °C
?. [Recombinant Plasmid Preparation and Verification with Restriction Digests]
Plasmid DNA extraction. Isolate the plasmid DNA from the cultures by using the GenElute™ PCR Clean-Up Kit according to the manufacturer’s instructions.
?. [Recombinant Plasmid Preparation and Verification with Restriction Digests]
VectorFD Enzyme(s)Empty vector fragment(s) (bp)Donor plasmid (Zero Blunt™ TOPO™)EcoRI3501, 18GGDest1-AmpEcoRI, BcuI (SpeI)3245, 57GGDest2-AmpBamHI, HindIII3243, 36GGDest3-AmpNdeI, LguI (SapI)2936, 314, 34GGDestX1-AmpNdeI, XhoI2523, 707GGDestX2-AmpEcoRI, XbaI3433, 856ReagentVolume per Miniprep (µL)Master MixFinal ConcentrationMiniprep DNA2.00(50 - 500 ng)FastDigest Green Buffer (10X)1.51.5 x (n + 1)1X*Enzyme(s)2.01.0 x (n + 1)MB H2O10.510.5 x (n + 1)Total15.013.0 x (n + 1)
13 µl
2 µl
37 °C
VectorFD Enzyme(s)Empty vector fragment(s) (bp)Donor plasmid (Zero Blunt™ TOPO™)EcoRI3501, 18GGDest1-AmpEcoRI, BcuI (SpeI)3245, 57GGDest2-AmpBamHI, HindIII3243, 36GGDest3-AmpNdeI, LguI (SapI)2936, 314, 34GGDestX1-AmpNdeI, XhoI2523, 707GGDestX2-AmpEcoRI, XbaI3433, 856
ReagentVolume per Miniprep (µL)Master MixFinal ConcentrationMiniprep DNA2.00(50 - 500 ng)FastDigest Green Buffer (10X)1.51.5 x (n + 1)1X*Enzyme(s)2.01.0 x (n + 1)MB H2O10.510.5 x (n + 1)Total15.013.0 x (n + 1)
?. [Recombinant Plasmid Preparation and Verification with Restriction Digests]
Gel electrophoresis of digested DNA. After the restriction digest is complete, run a sample of the digest on a gel to verify the length of the sequence ligated into the vector. Cast a 1% (wt/vol) agarose gel in TAE buffer with SYBR DNA gel stain. Load  of GeneRuler 1 kb DNA ladder into the first well and  of digest product into the next wells. Run the gel at 100 V for . Successful ligation of the correct insert sequence into the donor vector should yield a length of the full length insert plus the distance of the enzyme cut sites to the insert.
3 µl
15 µl
?. [Recombinant Plasmid Preparation and Verification with Restriction Digests]
Validation of plasmids via Sanger Sequencing. Verify the sequence of the isolated plasmids by sequencing using the primer pair appropriate for the vector (Table 2). The resulting sequences should be compared to a reference sequence for the expected plasmid product.
?. [Golden Gate Assembly of Fusion Protein Plasmids]
Preparation of donors and destination vector for single-pot assembly. The Golden Gate cloning method for single-pot assembly requires a 1:1 molar ratio of each insert donor to the destination vector, with a recommended amount of 1 μl each for the reaction.
?. [Golden Gate Assembly of Fusion Protein Plasmids]
Use a spectrophotometer to measure the concentrations (ng/µl) of donor and destination vector DNA stock.
?. [Golden Gate Assembly of Fusion Protein Plasmids]
Determine the starting molarity of each stock DNA:dsDNA fmol/µL = (dsDNA ng/µl / ((length of dsDNA bp x 617.96 ng/nmol/bp) + 36.04 ng/nmol) ) / 10^6Note: New England Biolabs provides a tool, the NEBioCalculator (https://nebiocalculator.neb.com/#!/dsdnaamt), for converting DNA length to mass.
?. [Golden Gate Assembly of Fusion Protein Plasmids]
SampleStock DNAWorking solution: DNAWorking solution: H2OLength (bp)ng/µLCalculated fmol/µLDNA µLMB H20 µLDonor A20050404.54.945.1Donor B20020016181.248.8Donor C10005080.9124.725.3Donor D1000200323.66.243.8Destination Vector3500300138.714.435.6SampleStock DNAWorking solution: DNAWorking solution: H2OLength (bp)ng/µLCalculated fmol/µLDNA µLMB H20 µLDonor A20050404.54.945.1Donor B20020016181.248.8Donor C10005080.9124.725.3Donor D1000200323.66.243.8Destination Vector3500300138.714.435.6
50 µl
SampleStock DNAWorking solution: DNAWorking solution: H2OLength (bp)ng/µLCalculated fmol/µLDNA µLMB H20 µLDonor A20050404.54.945.1Donor B20020016181.248.8Donor C10005080.9124.725.3Donor D1000200323.66.243.8Destination Vector3500300138.714.435.6
SampleStock DNAWorking solution: DNAWorking solution: H2OLength (bp)ng/µLCalculated fmol/µLDNA µLMB H20 µLDonor A20050404.54.945.1Donor B20020016181.248.8Donor C10005080.9124.725.3Donor D1000200323.66.243.8Destination Vector3500300138.714.435.6
?. [Golden Gate Assembly of Fusion Protein Plasmids]
Golden Gate reaction set-up. Prepare the reaction(s) according to the table below, with a final volume of  per reaction.ReagentVolume (µL)Final Amount or ConcentrationDonor A1.040 fmolDonor B1.040 fmolDonor C1.040 fmolDonor D1.040 fmolDestination Vector1.040 fmolT4 DNA Ligase Buffer with 10 mM ATP (10X)1.01X Buffer, 1 mM ATP*Quick Ligase (T4)0.5FastDigest BpiI (BbsI)0.5MB H2O3.0Total10
10 µl
ReagentVolume (µL)Final Amount or ConcentrationDonor A1.040 fmolDonor B1.040 fmolDonor C1.040 fmolDonor D1.040 fmolDestination Vector1.040 fmolT4 DNA Ligase Buffer with 10 mM ATP (10X)1.01X Buffer, 1 mM ATP*Quick Ligase (T4)0.5FastDigest BpiI (BbsI)0.5MB H2O3.0Total10
?. [Golden Gate Assembly of Fusion Protein Plasmids]
Golden Gate assembly reaction. Run the single-pot assembly reaction with the following thermocycler program:Cycle NumberDigestionLigationHeat Inactivation1-2545°C, 2 min16°C, 5 min2660°C, 10 min2780°C, 20 min
Cycle NumberDigestionLigationHeat Inactivation1-2545°C, 2 min16°C, 5 min2660°C, 10 min2780°C, 20 min
?. [Golden Gate Assembly of Fusion Protein Plasmids]
Quick transformation. Transform the Golden Gate reaction product into a competent E. coli strain, using guidelines appropriate for the strain. We recommend NEB DH5ɑ-Turbo. Quick transformation (skipping outgrowth in SOC) is possible for plasmids that carry an ampicillin resistance marker, i.e. the GGDest series described in the guidelines.
?. [Synthetic Chromatin Protein Expression in vitro for GGDestX1-Amp Plasmids]
The following procedure is for fusion proteins that include a fluorescent protein tag and have been constructed in GGDestX1-Amp, which carries an Or2-Or1-Pr promoter that enables strong expression in cell-free reactions [53]. For non-fluorescent proteins, additional guidelines for a Bradford protein quantification assay are provided.Prepare DNA templates. Prepare working solutions of 45 ng/µL for the recombinant plasmid (built from GGDestX1-Amp) and a GGDestX1-Amp (or P70a(2)-deGFP) control. The expected yield for preparations of P70a, GGDestX1-Amp and their derivatives is low because the backbone carries a ColE1 origin of replication (15 - 20 plasmid copies per cell).
?. [Recombinant Plasmid Screening with Colony PCR]
Label and pre-warm an agar plate. Label the bottom surface of an LB agar plate (with the appropriate selection antibiotic) with a numbered grid (labeled 1, 2, 3 … n) to accommodate streaks for all colonies that will be screened. Incubate the plate at  until you are ready to use it at Step 11.3.
37 °C
?. [Recombinant Plasmid Screening with Colony PCR]
Pipette  of PCR Master Mix into one PCR tube per colony (labeled 1, 2, 3 … n).
25 µl
?. [Recombinant Plasmid Screening with Colony PCR]
Pick a colony from the ligation plate with a clean sterile micropipette tip or inoculation needle, streak the colony on the agar plate in numbered square within the grid, and gently swirl the same tip or inoculation needle in the corresponding PCR tube with Master Mix.
?. [Recombinant Plasmid Screening with Colony PCR]
Run the following thermocycler program.Cycle NumberDenatureAnnealExtend195°C, 5 min2-3695°C, 15 s*#°C, 15 s72°C, 30 s3772°C, 3 min
Cycle NumberDenatureAnnealExtend195°C, 5 min2-3695°C, 15 s*#°C, 15 s72°C, 30 s3772°C, 3 min
?. [Generation of Plasmid Donors (optional)]
Pipette the  reaction product into a 2.0 mL microcentrifuge tube and add  chemically competent DH5ɑ-T cells. Gently tap the tube three times and incubate on ice for .
6 µl
50 µl
?. [Generation of Plasmid Donors (optional)]
Heat shock at  for .
42 °C
?. [Generation of Plasmid Donors (optional)]
Add  SOC outgrowth medium and incubate on a shaking platform at .
1 mL
1 33
?. [Generation of Plasmid Donors (optional)]
Pellet the cells at  and discard the supernatant.
Centrifuge: 16300 34, 3 min, 25 32
?. [Generation of Plasmid Donors (optional)]
Resuspend the pellet(s) in  50 µg/mL kanamycin LB broth. Plate each resuspension onto a pre-warmed 50 µg/mL kanamycin LB agar plate and spread using beads. Incubate at .
50 µl
37 °C
?. [Golden Gate Assembly of Fusion Protein Plasmids]
Plasmid screening with colony PCR. Follow the steps described under “Recombinant Plasmid Screening With Colony PCR” to identify 4 - 6 candidate clones with full-length inserts.
?. [Golden Gate Assembly of Fusion Protein Plasmids]
Recombinant plasmid preparation and verification. Follow the steps described under “Recombinant Plasmid Preparation and Verification with Restriction Digests.” It is important to include Sanger Sequencing (one forward reaction and one reverse reaction per construct) to determine if modules A - D were assembled in the correct order.
?. [Customization of Target-Specific gRNA in GGDestX2-Amp plasmids]
Design and produce gRNA dsOligos. If you used GGDestX2-Amp to build a dCas9 fusion protein in the previous steps, use the following procedure to customize the guide RNA sequence.
?. [Customization of Target-Specific gRNA in GGDestX2-Amp plasmids]
Design ssOligos according to Table 4 below to produce a double-stranded oligo that is specific to the 20 bp genomic target site of interest and has single-stranded overhangs that are compatible with the BsaI-generated ends in GGDestX2-Amp. ssOligossOligo Sequence (5’-3’)DescriptiongRNA topCACCNNNNNNNNNNNNNNNNNNNNCACC = BsaI overhang, N20 = gRNA target sequencegRNA bottomAAACNNNNNNNNNNNNNNNNNNNNAAAC = BsaI overhang, N20 = reverse complement of target sequence
ssOligossOligo Sequence (5’-3’)DescriptiongRNA topCACCNNNNNNNNNNNNNNNNNNNNCACC = BsaI overhang, N20 = gRNA target sequencegRNA bottomAAACNNNNNNNNNNNNNNNNNNNNAAAC = BsaI overhang, N20 = reverse complement of target sequence
?. [Customization of Target-Specific gRNA in GGDestX2-Amp plasmids]
DsOligo drop-in reaction set-up. After you have verified a GGDestX2-Amp clone that has a properly assembled dCas9 fusion fragment, prepare Type IIS digestion T7 ligation reaction(s) as shown in the table below, with a final volume of  per reaction. ReagentVolume (µL)Final Amount or ConcentrationRecombinant GGDestX2-Amp plasmid≤ 6.3 µL50 ng dsOligo (1X) (40 nM)1.04 nM dsOligoFermentas Buffer G (10X)1.01X Fermentas Buffer G10 mM DTT0.50.5 mM DTT10 mM ATP0.50.5 mM ATPBsaI (Eco31I)0.5T7 DNA ligase0.2MB H2OBring final volume to 10 µLTotal10
10 µl
ReagentVolume (µL)Final Amount or ConcentrationRecombinant GGDestX2-Amp plasmid≤ 6.3 µL50 ng dsOligo (1X) (40 nM)1.04 nM dsOligoFermentas Buffer G (10X)1.01X Fermentas Buffer G10 mM DTT0.50.5 mM DTT10 mM ATP0.50.5 mM ATPBsaI (Eco31I)0.5T7 DNA ligase0.2MB H2OBring final volume to 10 µLTotal10
?. [Customization of Target-Specific gRNA in GGDestX2-Amp plasmids]
DsOligo drop-in reaction. Run the digestion-ligation reaction(s) with the following thermocycler program:Cycle NumberDigestionLigationHold1-637°C, 5 min23°C, 5 min74°C, ∞
Cycle NumberDigestionLigationHold1-637°C, 5 min23°C, 5 min74°C, ∞
?. [Customization of Target-Specific gRNA in GGDestX2-Amp plasmids]
PlasmidSafe treatment. Treat ligations with PlasmidSafe exonuclease to prevent unwanted recombination products. Incubate at  for .ReagentVolume (µL)Final Amount or ConcentrationDigestion-ligation reaction3.7PlasmidSafe Buffer (10X)0.51X PlasmidSafe Buffer10 mM ATP0.51 mM ATPPlasmidSafe exonuclease0.3Total5
37 °C
ReagentVolume (µL)Final Amount or ConcentrationDigestion-ligation reaction3.7PlasmidSafe Buffer (10X)0.51X PlasmidSafe Buffer10 mM ATP0.51 mM ATPPlasmidSafe exonuclease0.3Total5
?. [Customization of Target-Specific gRNA in GGDestX2-Amp plasmids]
Transformation. Transform  of chemically competent E. coli (e.g. DH5ɑ-Turbo) with all  of the PlasmidSafe reaction. Follow the quick transformation procedure described in Steps 22.1 - 22.2.
50 µl
5 µl
?. [Customization of Target-Specific gRNA in GGDestX2-Amp plasmids]
Recombinant Plasmid Preparation and Verification. Follow the steps described under “Recombinant Plasmid Preparation and Verification with Restriction Digests” to extract and purify plasmid DNA from 2 - 4 colonies, but do not perform the restriction digest. Use Sanger Sequencing (U6 primer, Table 2) to determine if the gRNA region contains the appropriate custom target sequence. Although the background colony count (from a vector-only digestion-ligation control) is usually high, the gRNA drop-in is extremely efficient. In our hands a desired clone can often be found by picking just one or two colonies.
?. [Synthetic Chromatin Protein Expression in vitro for GGDestX1-Amp Plasmids]
TXTL reaction set-up. Prepare reactions in a 96-well qPCR plate. Test each plasmid at 62.5 and 125.0 ng/well at minimum. Test additional dilutions when comparing rates of expression over several template concentrations [53]. Set up each unique reaction in triplicate wells by first adding the template DNA (≤ ), the amount of MB H2O needed to bring the final reaction to , then the myTXTL reagent. Include at least one mock (negative control) well. A single mock control well is sufficient if the amount of myTXTL reagent is in limited supply.ReagentVolume (µL)125 ng DNA62.5 ng DNA0 ng* (mock)myTXTL reaction buffer999Template DNA (45 ng/µL)2.81.40MB H2O0.21.63Total121212
3 µl
12 µl
ReagentVolume (µL)125 ng DNA62.5 ng DNA0 ng* (mock)myTXTL reaction buffer999Template DNA (45 ng/µL)2.81.40MB H2O0.21.63Total121212
?. [Synthetic Chromatin Protein Expression in vitro for GGDestX1-Amp Plasmids]
TXTL reactions in a real-time thermocycler. Run the TXTL reaction for 96 10-min cycles at . For each cycle, include a signal detection step (record) to measure expression of the recombinant plasmids. Use a dye-detection channel that is appropriate for the type of fluorescent protein that is encoded in the plasmid (for examples see the table below).CycleHoldRecord1-9629°C, 9:59 min29°C, 1 secFluorescent proteinEmission (nm)Dye detection channelEmission (nm)EGFP (P70a or GGDestX1-Amp)509*SYBR520EGFP (P70a or GGDestX1-Amp)509FAM517AmCyan491FAM517Venus528SYBR520mCherry610*Texas Red604mCherry610ROX603
29 °C
CycleHoldRecord1-9629°C, 9:59 min29°C, 1 sec
Fluorescent proteinEmission (nm)Dye detection channelEmission (nm)EGFP (P70a or GGDestX1-Amp)509*SYBR520EGFP (P70a or GGDestX1-Amp)509FAM517AmCyan491FAM517Venus528SYBR520mCherry610*Texas Red604mCherry610ROX603
?. [Synthetic Chromatin Protein Expression in vitro for GGDestX1-Amp Plasmids]
Data analysis. Download the raw data (time versus fluorescence signal) from the real time thermocycler. Use software (e.g. Microsoft Excel or GraphPad Prism) to plot time on the x-axis and mean fluorescent signal and standard deviation on the y-axis. For each series determine the (1) maximum output signal and the (2) maximum protein production rate (slope of the trend if linear, Hill slope if nonlinear, see Fig. 6).
?. [Synthetic Chromatin Protein Expression in vitro for GGDestX1-Amp Plasmids]
Bradford assay. A Bradford assay, or similar protein quantitation assay can be used to support the results from fluorescent signal detection, or if the synthetic chromatin fusion protein has no detectable fluorescence.
?. [Customization of Target-Specific gRNA in GGDestX2-Amp plasmids]
Follow the guidelines from Ran et al. to identify a 20 bp target site near the gene or within a genomic region of interest [52]. We recommend the Benchling CRISPR design tool (https://www.benchling.com/crispr/) for target detection and scoring.
?. [Synthetic Chromatin Protein Expression in vitro for GGDestX1-Amp Plasmids]
Prepare the following samples in a final volume of  in MB H2O: background ( MB H2O), BSA standards (0.125, 0.25, 0.5, 0.75, 1.0, 1.5, and 2.0 mg/mL), experimental TXTL samples (10 - 100% v/v), and mock TXTL (10 - 100% v/v).
5 µl
5 µl
?. [Synthetic Chromatin Protein Expression in vitro for GGDestX1-Amp Plasmids]
Combine  diluted sample and  Bradford reagent in each well of a microwell plate. Set up triplicate wells for each sample where possible. We recommend testing diluted TXTL samples first and then increasing the concentration as needed.
5 µl
250 µl
?. [Synthetic Chromatin Protein Expression in vitro for GGDestX1-Amp Plasmids]
Incubate at room temperature for  and measure the absorbance at 595 nm in a microwell plate reader.
?. [Synthetic Chromatin Protein Expression in vitro for GGDestX1-Amp Plasmids]
Create a standard curve and linear regression using background-subtracted BSA standard values. Subtract the mock TXTL signal from the experimental TXTL values and use the equation from the linear regression to determine the protein concentration for each experimental sample.
?. [Recombinant Plasmid Screening with Colony PCR]
Remove the gel from the electrophoresis chamber and use an imager to record the results. Compare the observed results to the expected length(s) of the PCR products. Positive clones should yield a single product the length of the donor fragment plus the distance of the primer to the cloning site.
?. [Recombinant Plasmid Screening with Colony PCR]
Place the streak plate from Step 11.3 in a  incubator . The next day, seal the plate with Parafilm and store it at  . Bacteria from streaks for confirmed clones will be used to inoculate new liquid cultures later.
37 °C
4 °C
?. [Golden Gate Assembly of Fusion Protein Plasmids]
Combine  of the product with  of chemically competent cells and let incubate on ice for .
5 µl
50 µl
?. [Golden Gate Assembly of Fusion Protein Plasmids]
Pipette the transformed cells onto a warmed agar plate (100 μg/mL ampicillin). Spread the cells with plating beads and place in a  incubator for at least  for DH5ɑ-Turbo cells. Other strains may require overnight incubation.
37 °C
?. [Customization of Target-Specific gRNA in GGDestX2-Amp plasmids]
Set up reactions for phosphorylation and annealing of ssOligos as shown in the table below. ReagentVolume (µL)Final Amount or Concentration100 µM gRNA top ssOligo1.010 µM ssOligo100 µM gRNA bottom ssOligo1.010 µM ssOligoT4 ligation buffer (10X)1.01X T4 ligation bufferT4 PNK0.3MB H2O6.7Total10250X dsOligo (10 µM)
ReagentVolume (µL)Final Amount or Concentration100 µM gRNA top ssOligo1.010 µM ssOligo100 µM gRNA bottom ssOligo1.010 µM ssOligoT4 ligation buffer (10X)1.01X T4 ligation bufferT4 PNK0.3MB H2O6.7Total10250X dsOligo (10 µM)
?. [Customization of Target-Specific gRNA in GGDestX2-Amp plasmids]
Run the ssOligo phosphorylation and annealing reaction(s) with the thermocycler program shown in the table below. Dilute   of the resulting 250X dsOligo solution in   MB H2O to make a 1X working solution for Step 26.  Cycle NumberPhosphorylationPNK Deactivation & DNA meltingDNA AnnealingHold137°C, 30 min295°C, 5 min3Ramp down at 5°C per min to 25°C44°C, ∞
1 µl
249 µl
Cycle NumberPhosphorylationPNK Deactivation & DNA meltingDNA AnnealingHold137°C, 30 min295°C, 5 min3Ramp down at 5°C per min to 25°C44°C, ∞