Editing Join Fragments by Taq Ligase

[[File:SOC.png|RTENOTITLE]]

[[File:SOC2.png|RTENOTITLE]]

[[File:SOC3.png|RTENOTITLE]]

[[File:SOC4.png|RTENOTITLE]]

Gibson Assemblies were designed for megabase-sized DNA sequences with hundreds of base pairs of homology, but have been adapted to the kilobase and smaller scale of common laboratory applications such as protein engineering [9]. Gibson Assembly has limitations too, such as increasing inefficiency when the number of inserts increases, inability to assembly small (<100 bp) sequences, and complex and error-prone addition of homologous sequences that are needed to direct the orientation and order of the assembled product.

'''Primer Phosphorylation'''

{|
|-
| Component
| Amount (µl)
| Final Concentration
|-
| 200 µM Forward Primer
| 0.75
| 3 µM
|-
| 200 µM Reverse Primer
| 0.75
| 3 µM
|-
| 10 U/µl T4 Polynucleotide Kinase
| 1
| 0.2 U
|-
| 10X T4 Ligase Buffer
| 5
| 1X
|-
| H<sub>2</sub>O
| 42.5
| <br/>
|-
| Total
| 50
| <br/>
|-
| colspan="3" | Incubation: 37ºC for 30 Minutes&nbsp;<br/>
|}

'''Cycled Ligation Reaction'''

Linearized vector was diluted in H2O to a concentration of 5 nM. The desired inserts for a specific cycled ligation assembly reaction were mixed and diluted in H2O to form an insert mixture with a concentration of 20 nM for each insert. 1 µl of each scaffold oligonucleotide connector (200 µM) used in a specific cycled-ligation assembly reaction was added into 100 µl of H2O, and this SOC mixture was further diluted 1∶10 in H2O (200 nM) before use. For Cycled Ligation Assemblies without a vector, the volume of the linearized vector was replaced with H2O.

SOC: 40 bp Scaffold Oligonucleotide Connectors

Assembly of fragment:&nbsp;fragments from <500 bp to 5 kb

{| border="0" cellspacing="0" cellpadding="0"
|-
| Component
| Amount (µl)
| Final Concentration
|-
| 5 nM Linearized pUC19
| 4
| 1 nM
|-
| 20 nM Insert Mix
| 2
| 2 nM
|-
| 200 nM SOC Mix
| 1
| 10 nM
|-
| 10X Taq Ligase Buffer
| 2
| 1X
|-
| 40 U/µl Taq DNA Ligase
| 2
| 80 U
|-
| H<sub>2</sub>O
| 9
| &nbsp;
|-
| Total
| 20
| &nbsp;
|-
| colspan="3" | Thermocycler Program:
|-
| colspan="3" | 1. 95ºC 2 Min
|-
| colspan="3" | 2. 95ºC 30 Sec
|-
| colspan="3" | 3. 60ºC 2 Min
|-
| colspan="3" | 4. GOTO 2 30x
|-
| colspan="3" | 5. 55ºC 10 Min
|-
| colspan="3" | 6. 4ºC HOLD
|}

When using CLAs to directly assemble inserts into a plasmid, the plasmid must be linearized using'''blunt ended restriction digestions'''&nbsp;such as NruI, SwaI due to non-productive re-ligation of sticky ends by Taq ligase.&nbsp;Gibson Assemblies do not have this limitation&nbsp;due to its lower Taq ligase concentrations. Golden Gate Assemblies are better suited for high throughput applications than CLAs or Gibson assemblies

'''DNA Insert PCR Amplification'''
<div>
{| border="0" cellspacing="0" cellpadding="0"
|-
| Component
| Amount (µl)
| Final Concentration
|-
| Primer Phosphorylation Mix
| 4
| 0.5 µM each (Fwd, Rev)
|-
| 10 ng/µl Template Plasmid
| 1
| 10 ng
|-
| 2.5 mM dNTPs
| 2
| 200 µM
|-
| 5X Phusion HF Buffer
| 5
| 1X
|-
| 2 U/µl Phusion Polymerase
| 0.25
| 0.5 U
|-
| H<sub>2</sub>O
| 12.75
|-
| Total
| 25
|-
| colspan="3" | Thermocycler Program:
|-
| colspan="3" | 1. 98ºC 30 Sec
|-
| colspan="3" | 2. 98ºC 5 Sec
|-
| colspan="3" | 3. 55ºC 15 Sec
|-
| colspan="3" | 4. 72ºC X Sec
|-
| colspan="3" | 5. GOTO 2 30x
|-
| colspan="3" | 6. 72ºC 5 Min
|-
| colspan="3" | 7. 4ºC HOLD
|-
| colspan="3" | Extension Time X = Product Size (kb) * 30, Minimum 30 sec
|}
</div><div><br/></div><div></div>
'''Double Blunt Vector Digestion'''<br/>ComponentAmount (µl)Final Concentration<br/>100 ng/µl pUC19 Vector5500 ng<br/>10 X NEB Buffer 351X<br/>100 X BSA0.51X<br/>10 U/µl NruI110 U<br/>10 U/µl SwaI110 U<br/>H2O37.5<br/>Total50<br/>Incubation: 37ºC for 1 Hr

== <br/>TPA ==

'''Design of TPA oligonucleotides'''<br/>For each DNA fragment to be assembled, four oligonucleotides were ordered: long forward (LF), short forward (SF), long reverse (LR) and short reverse (SR). The long primers (LF and LR) were analogous to that used in GA, and they defined the overlap region, whereas the short primers stopped exactly before the overlap. The TPA oligonucleotides design algorithm was as follows:

The melting temperature (TM) of the overlap region (Figure (Figure1B)1B) was set at 50°C as predicted by IDT's OligoAnalyzer using the following modified conditions: Oligo concentration = 0.015 μM, Na+ = 50 mM, Mg2+ = 10 mM. All junctions used in an assembly should have the same TM.

<br/>As much as possible, G/C locks (i.e. the nucleotide closest to the nick being a G or C) were used at both sides of the nicks when choosing junction sequences. In situations where such locks were not possible, consecutive T/A close to the nicks should be avoided.

<br/>The overlap regions of the long primers were defined by their TM rather than by length. Long primers with TM = 50°C junctions were usually GA compatible.<br/>The short primers (SF or SR) were ordinary PCR primers adjacent to the overlap sequences.

<br/>'''Two-step assembly: preparation of fragments'''<br/>For each DNA fragment to be assembled, two PCR products were generated: one with primers LF and SR, the other with primers SF and LR. PCR reactions were performed using either KOD Xtreme Hotstart (Merck, Darmstadt, Germany) or Q5 (NEB, Ipswich, MA, USA) DNA polymerase according to the respective manufacturer's recommended protocol. Depending on PCR purity, either gel purification (QIAquick Gel Extraction Kit, Qiagen, Hilden, Germany) or PCR purification (QIAquick PCR Purification Kit, Qiagen, Hilden, Germany) were used to clean up the products. The two PCR products for each fragment were mixed in 1:1 molar ratio, to a final concentration of 40 fmol/μl DNA (40 fmol/μl ≈ 24 ng/kb/μl DNA) in 1× CutSmart buffer (NEB, Ipswich, MA, USA). A lower final concentration (down to 10 fmol/μl) was used for some fragments when the higher final concentration could not be achieved due to low PCR yield. An excel worksheet was used to facilitate the calculations (see Supplementary Protocol for details). If plasmid template was used in the PCR, 0.5 μl DpnI was added to eliminate the plasmid template so as to reduce the transformation background. The re-annealing step that generates overhangs was carried out using the following temperature profile: 37°C for 30 min (for DpnI digest), 98°C for 2 min, 85°C for 2 min (at a ramping rate of 0.1°C/s), 75°C for 2 min (0.1°C/s), 65°C for 2 min (0.1°C/s), 55°C for 2 min (0.1°C/s) and 8°C hold (0.1°C /s). The annealed DNA fragments were stored at 4°C until the assembly step.

'''Two-step assembly: assembly of fragments'''<br/>The assembly was carried out by hybridizing the re-annealed fragments. All the re-annealed DNA fragments were mixed in 1:1 molar ratio, to a final concentration of 4 fmol/μl/fragment (4 fmol/μl ≈ 2.4 ng/kb/μl) in 1× CutSmart buffer. The hybridization step that generates nicked circular plasmid was carried out using the following temperature profile: 65°C (TM + 15) for 10 s, 55.5°C (TM + 5.5) for 1–2 h and 8°C hold (0.1°C/s). The hybridized nicked plasmid was stored at 4°C until E. coli transformation.

'''One-step assembly'''<br/>In one-step assembly, all PCR products for the construct were put together in one-pot at a final concentration of 2 fmol/μl/product in 1× CutSmart buffer, 0.5 μl DpnI was added if necessary. The one-step assembly was carried out using the following temperature profile: 37°C for 30 min (DpnI digest), 98°C for 2 min, 85°C for 2 min (0.1°C/s), 75°C for 2 min (0.1°C /s), 65°C for 2 min (0.1°C/s), 55.5°C (TM + 5.5) for 1–2 h (0.1°C/s) and 8°C hold (0.1°C/s). The hybridized nicked plasmid was stored at 4°C until E. coli transformation.

'''E. coli transformation'''

Use E coli competent cells by chemical method for transformation

== [[File:TPA.png]] ==
Number of fragments (fidelity)Enzyme requiredReference


{|
|-
| colspan="4" | Ligase-independent assembly methods
|-
| CPEC
| 5 fragments for a 9 kb plasmid (90%)
| DNA polymerase
| [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499748/#B17 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499748/#B17]
|-
| In-fusion
| 5 fragments for a 5 kb plasmid (90%)
| In-fusion HD enzyme mix
| [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499748/#B18 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499748/#B18]
|-
| Hot fusion
| 8 fragments for a 8 kb plasmid (90%)
| T5 Exonuclease, DNA polymerase
| [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499748/#B19 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499748/#B19]
|-
| MOE-PCR
| 7 fragments for a 7 kb plasmid (25%)
| DNA polymerase
| [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499748/#B22 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499748/#B22]
|-
| SLICa
| 10 fragments for a 8 kb plasmid (20%)
| T4 DNA polymerase, RecA
| [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499748/#B20 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499748/#B20]
|-
| SLiCEa
| 7 fragments for a 5 kb plasmid (90%)
| Escherichia coli&nbsp;extract
| [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499748/#B21 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499748/#B21]
|-
| USER
| 8 fragments for a 11 kb plasmid (60%)
| USER enzyme mix
| [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499748/#B10 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499748/#B10]
|-
|-
| colspan="4" | Enzyme-independent assembly methods
|-
| EFC
| 2 fragments for a 3 kb plasmid (90%)
| 
| [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499748/#B23 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499748/#B23]
|-
| PIPE
| 2 fragments for a 5 kb plasmid (90%)
| 
| [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499748/#B13 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499748/#B13]
|-
| SLICb
| 2 fragments for a 15 kb plasmid
| 
| [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499748/#B20 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499748/#B20]
|-
| TPAa
| 10 fragments for a 7 kb plasmid (80%)
| 
| https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499748/#B13
|}

The largest demonstrated number of fragments is shown for each method with the corresponding fidelity in bracket.

aThe largest demonstrated assembly sizes are as indicated except for SLIC (12 kb from 2 fragments), SLiCE (24 kb from 2 fragments) and TPA (31 kb from 5 fragments).

bSLIC has an enzyme independent protocol.







'''PIPE cloning'''<br/>0.025 pmol of vector and 0.0625 pmol insert purified products (2.5∶1 insert∶vector ratio) were digested for 3 hr at 37°C with 10 U of DpnI in 10 µL CutSmart Buffer (NEBuffer 4.1/0.1 mg/mL BSA), diluted 1∶1 with 1× HF buffer (to control for buffer composition between purified and unpurified products, as 1× HF buffer halved transformation efficiency), and 2 µL used to transform 18 µL of XL10 Gold ultracompetent cells (Agilent Technologies) according to manufacturer's instructions.

'''SLIC'''<br/>DpnI digested purified PIPE products were incubated at 25°C for 5 min with 0.75 U of T4 DNA polymerase, immediately placed on ice for 10 min, diluted 1∶1 with ice-cold 1× HF Buffer, and 2 µL used for transformation as described above.

'''OEC'''<br/>20 µL OEC reactions were performed using 10–250 fmol of insert product −84 bp: 250 fmol (12.5 nM); 350 bp: 100 fmol (5 nM); 1.4 kb: 25 fmol (1.25 nM); 4.3 kb: 10 fmol (0.5 nM) - as megaprimer and 25 ng of pUC18/Kan vector template with 5% DMSO, 0.4 U of Phusion Hot Start II High Fidelity DNA polymerase, and cycling conditions: 72°C 5 min (to blunt the megaprimer), 98°C 3 min, (98°C 30 s 63°C 30 s 72°C 3 min) ×30. 5 µL fractions were diluted 1∶1 with CutSmart Buffer (to control for buffer and assist DpnI activity), digested for 3 hr at 37°C with 20 U of DpnI, and 2 µL used for transformation as described above.
[[Category:Antibodies]] [[Category:Protocols]]