Constructing plasmid under GT DNA assembly standard CURRENT

We reported a new DNA assembly standard (GT standard, GTS), which allowed constructing plasmid using standard DNA parts and various DNA assembly methods in a near-scarless manner Here we provide a protocol to detail the experimental procedures to construct plasmid under GTS using CLIVA, Gibson assembly, In-fusion cloning, and restriction enzyme (RE)-based methods. DNA assembly is the cornerstone of genetic engineering 1 . Researchers are currently using various DNA assembly methods to construct plasmid by assembling multiple DNA parts 1 . We reported GT standard (GTS) 2 for plasmid construction under which DNA sequences are defined as two types of standard, reusable parts (fragment and barcode). We developed a barcoding method that can efficiently add any two barcodes to two ends of any fragment without leaving scars in most cases. In brief, the DNA fragments can be standardized by generating two conserved one nucleotide (1-nt) sticky ends (SEs, “C” and “T”) at both ends, which can be ligated (barcoded) with a pair of barcoding oligos with compatible SEs (“G” and “A”). After barcoding, we can assemble up to seven such barcoded fragments through long overlapping sequences (15 to 20 bp) into one plasmid by using one of the existing DNA assembly methods, including CLIVA, Gibson assembly, In-fusion cloning, restriction enzyme (RE)-based methods and Yeast in vivo assembly. GTS provides an open and flexible architecture, allowing users to flexibly define sharable barcode sequence with or without biological functions, and select the appropriate DNA assembly methods based on their preference and the requirements of plasmid construction. To promote researchers to adopt GTS for plasmid construction, a detailed protocol is provided here.


Introduction
DNA assembly is the cornerstone of genetic engineering 1 . Researchers are currently using various DNA assembly methods to construct plasmid by assembling multiple DNA parts 1 . We reported GT standard (GTS) 2 for plasmid construction under which DNA sequences are defined as two types of standard, reusable parts (fragment and barcode). We developed a barcoding method that can efficiently add any two barcodes to two ends of any fragment without leaving scars in most cases. In brief, the DNA fragments can be standardized by generating two conserved one nucleotide (1-nt) sticky ends (SEs, "C" and "T") at both ends, which can be ligated (barcoded) with a pair of barcoding oligos with compatible SEs ("G" and "A"). After barcoding, we can assemble up to seven such barcoded fragments through long overlapping sequences (15 to 20 bp) into one plasmid by using one of the existing DNA assembly methods, including CLIVA, Gibson assembly, In-fusion cloning,  a) Install and run the App. A graphic interface containing two modules for designing fragment-and barcode-associated oligos will appear. b) Design fragment-related oligos: Input name and sequence of fragment (>= 35 bp), and click button "Click to design oligos for FRAGMENT". Remember to include the conversed G and T in the input sequence. If the fragment length is longer than 90 bp, Foligos with or without phosphorothioate (PS)-bonds will be designed and displayed on the right-side of the window. If the fragment length is less than 91 bp, Noligos (Non-modified fragment oligos) will be designed and displayed in addition to Foligos. Users can use button "Copy oligo info" to copy information of the designed oligos to clipboard of the desktop's operating system (so far only Microsoft Windows 10 has been tested), which can be pasted into a spreadsheet of Microsoft Excel. The software arranges the oligo information according to format requirement of the IDT ordering system, and it determines the synthesis scale automatically. Click button "Clear results" to reset the software for the next design. c) Design barcode-related oligos: Input name and sequence of barcode (20-80 bp), and click button "Click to design oligos for BARCODE". Two types of Boligos and Aoligos will be designed, and the oligo information will be displayed. The software automatically determines SE sequence. If it cannot find a suitable SE sequence, user can consider to extend the barcode sequence to enlarge the search space.
User can manually specify the SE sequence to overwrite the SE search step but it may result in lower assembly efficiency. Boligos designed here can be used with CLIVA, Gibson and In-fusion assembly method. Aoligos designed here are for the CLIVA method. User may manually modify the designed 5 Aoligos for Gibson and In-fusion method: remove the PS modifications and extend SE sequence to meet requirement of those methods. This version of the software does not support RE-based cloning and we will release new version to support them.
Step 2: Prepare GTS oligos a) All GTS oligos with or without PS bonds (dried form) purchased from oligo manufacturer should be dissolved in nuclease-free water to a final concentration of 100 µM, and can be stored at -20 °C for long-term use. 3) Twenty microliters of the phosphorylated and folded Boligos are diluted using 60 µL of nucleasefree water, and can be stored at -20 °C for long-term use.

Step 3: Prepare GTS fragments
User can choose one of the following three options to prepare a GTS fragment.

a) Creating fragment using PS-modified Foligos
Fragments can be amplified from various sources (e.g., plasmid, synthetic DNA, genomic DNA) using PS-modified Foligos. 7) The prepared fragment can be stored at -20 °C for long-term use. b) Creating fragment using non-modified Foligos Fragments can be amplified from various sources (e.g., plasmid, synthetic DNA, genomic DNA) using non-modified Foligos. 7 1) The steps of amplification of fragments using non-modified oligos are the same to Step 3a 1 and 3a 2.
2) The steps of isolation and purification of fragment are the same to Step 3a 3 and 3a 4. At the last step, use 30 µL of HPLC water to elute DNA fragments from column. c) Creating fragment using Noligos Short fragments can be directly created by annealing Noligos.
2) The annealing is done by the following program in a thermo cycler: 98 °C for 2 min 3) The fragments prepared by annealing Noligos are diluted with nuclease-free water to a final concentration of 10-20 ng/µL, and can be stored at -20 °C for long-term use.

Step 4: Ligate Boligos with GTS fragments
The ligation of two Boligos to two ends of a fragment is termed as barcoding. User can choose the following two options to barcode a fragment. Note: The recommended minimal fragment concentration is 10 ng/µL for fragment no longer than 1 kb. If fragment is larger than 1 kb, we recommend to use at least 100 ng/µL; if fragment is larger than 2 kb, we recommend to use at least 200 ng/µL. Vacufuge can be used to concentrate fragment solution when the concentration is too low. It is critical to have high purity of fragment with 1-nt SEs, so absorption spectrum (200-300 nm) of each fragment solution should be examined by Nanodrop or a similar device to ensure there is a peak at 260 nm before its fragment concentration can be used in the calculation.
2) The barcoding reaction is done using the following program in a thermo cycler: 25 °C for 5 min, 8 and hold at 4 °C. b) Barcoding fragment prepared using non-modified oligos A one-pot reaction is performed to generate the fragment with 1-nt SEs using non-modified oligos. 2) The mixture is incubated at 37 °C for 1 hour, 25 °C for 10 min, and hold at 4 °C.
Step 2) PCR cycling condition is the same to Step 3a 2.
3) The isolation of gel containing appropriate bands after gel electrophoresis is the same to Step 3a 3.

4) The purification of gel containing appropriate bands is the same to
Step 3a 4. In the end, use 40 µL ultrapure water to elute the column. 2) The other steps are same to the Step 3a 2 to Step 3a 4.
Step 6: Prepare GTS barcoded fragment a) If the barcoded fragments are prepared using non-modified Aoligos, 40 µL of barcoded fragment prepared in Step 5a 4 and 5b 2 can be directly used in DNA assembly step using Gibson or In-fusion cloning method. b) If RE-based method is used, a barcoded fragment will be amplified using non-modified Aoligos containing desired RE sites. The barcoded fragment prepared in Step 5a 4 or 5b 2 will be enzymatically treated to generate SEs, and then the treated fragment can be ligated with a plasmid digested using the same REs.
1) The RE-digestion solution contains 1 µL of RE1 and 1 µL of RE2 that recognize two cutting sites on both ends of barcoded fragment, 1 µg of barcoded fragment, 5 µL of 10X proper Buffer, and nucleasefree water to 50 µL. The users should follow general rules of REs here, such as avoiding internal cutting sites.
2) The RE-digestion mixture is incubated at 37 °C for 3 hours (overnight can be used to achieve complete digestion), and the appropriate fragments are separated using gel electrophoresis, and then purified using column as described in Step 3a 4.
3) Twenty-five microliters of enzymatically treated barcoded fragment are typically obtained from the previous step. c) If barcoded fragment is amplified using PS-modified Aoligos, iodine-based cleavage reaction is performed to treat barcoded fragment (prepared in Step 5a 4 or 5b 2) to generate long SEs at its both ends as described in Step 3a 5. Thirty microliters of purified barcoded fragment can be obtained for subsequent assembling step using CLIVA method. 10 d) The concentrations (ng/µL) of the barcoded fragments (purified after chemical or enzymatic treatment) are determined using NanoDrop or any other similar device.

Step 7: Assemble GTS barcoded fragments
The barcoded fragments obtained in Step 6 are assembled into plasmid using various DNA assembly methods. a) Gibson method: 1) The assembly reaction solution contains 4 µL of 2X Gibson Assembly Master Mix, and 4 µL of fragment mixture containing the equimolar barcoded fragments obtained in Step 5d. The recommended quantity of total barcoded fragment is 0.2-1 pmols.
2) The assembly reaction is performed at 50 °C for 15 min (2 to 3 fragments) or 60 min (4 to 6 fragments) in a thermo cycler. Two microliter of the assembly mixture is used for transformation. 2) The assembly reaction is performed at 50 °C for 15 min in a thermo cycler. One microliter of the assembly mixture is used for transformation. c) RE-based method 1) The ligation solution contains 0.5 µL of T4 ligase, 0.5 µL of 10X T4 ligase buffer, 4 µL of mixture containing the enzymatically digested barcoded fragment and backbone obtained in Step 6b 3 (the molar ratio of insert to backbone is 3:1).
2) The assembly reaction is done at 16 °C for 12 h in a thermo cycler. One microliter of the assembly mixture is used for transformation. 2) The assembly reaction solution is heated in a thermo cycler at 80 °C for 1 min, decreased to 68°C at a default speed of thermal cycler, kept for 10 min and then decreased to 4 °C at 0.1 °C/s. One microliter of the assembly mixture is used for transformation.
Step 8: Transformation a) One microliter of assembly products obtained in Step 7a, 7b, 7c and 7d is mixed with 17 µL of E.
coli Dh5α heat-shock competent cell in a pre-chilled 1.7 mL Eppendorf microcentrifuge tube on ice for 5 min.
b) The tube is heat-shocked at 42 °C in an Eppendorf ThermoMixer for exactly 35 s, and then incubated on ice for 2 min. The cell solution is mixed with 150 µL of SOC medium and directly plated on LB Agar plate that contains proper antibiotic.
c) The plate is incubated at the temperature required by specific applications. Usually, colony appears after 12-16 h when incubated at 37 °C.
Step 9: Colony PCR verification a) E. coli colony PCR reaction solution contains 1 µL of colony suspension (single colony obtained in Step 8c is resuspended using 100 µL of ultrapure water in 0. c) The sequencing data received can be analysed using Benchling or similar software. The data with ab1 format are uploaded, and are aligned with plasmid sequence.
Notes: 1) One more plasmid can be sent out for sequencing if mutation/insertion/deletion that affects the biological function of plasmid is encountered in the sequenced one.
2) The sequencing results should cover the important regions of plasmid (e.g., barcode sequence with biological function [ribosomal binding site, 5'-and 3'-untranslated regions, protein linker, etc.] and coding sequence).

Troubleshooting
Step 3a Problem: Non-specific amplicons in amplification of fragment Possible reason: Low specificity of Foligos to template Solution: Run gel electrophoresis for longer time until a clear target band can be isolated from non-specific ones; Adjust temperature in annealing step of PCR to improve yield of target band.

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Step 3a Problem: Failed to amplify long fragment 2) Use and store enzymes properly by following general molecular biology practices.
Step 5a Problem: 1) Increase concentration of fragment with 1-nt SEs using Vacufuge; Increase PCR volume to 100 µL, and purify four slices of gel containing appropriate band using one column, and then elute DNA using 30 µL of nuclease-free water.
2) The quality of purified product should be checked using Nanodrop to ensure a clear peak is observed at A260 before barcoding.
3) Aliquot the reagents (DNA polymerase and ligase) in small volume for multiple time use, and place the reagents on ice during use.
Step 6b Problem: 14 Failed to digest fragments Possible reason: 1) The enzymatic reaction is not completed 2) Decay of reagents Solution: 1) Increase the incubation time of enzymatic reaction.
2) Aliquot the reagents (restriction enzymes) for multiple time use, and place the reagents on ice during use.
Step 8 Problem: Low assembly efficiency of plasmid (less colony appeared on the plate) 1) Try a different DNA assembly method.
2) Ensure appropriate molar ratio of barcoded fragments based on DNA assembly method.
3) Use inducible promoter or replication origin with low copy number to lower the expression of target gene that could be toxic to cell Step 10a Use 20 mL of cell culture to extract plasmid with low copy number.
Step 10c Problem: No signal of sequencing data 1) Use another primer that can efficiently bind to target region of plasmid.
2) Check all the fragments using colony PCR before sequencing to exclude the failed construction.
3) Culture the cells using fresh LB medium and appropriate amount of antibiotics.

Anticipated Results
All the results can be found in the paper 2 , named "A standard for near-scarless plasmid construction using reusable DNA parts".