Gibson Assembly
这个方法是在一个反应体系里面，一步合成多个片段的基因组装技术。
具体的原理是：
1. 核酸外切酶从5’端移除单个核苷酸，同源片段通过anneal（退火）搭在一起；
2. DNA聚合酶以某一链为模板，补充缺失的片段；
3. DNA连接酶形成磷酸二酯键，最后产生完整、没有连接缝隙的DNA分子。
It was found that overlapping DNA
molecules could be efficiently joined using three enzyme specificities:
(i)exonuclease activity, that chews back the ends of DNA fragments and exposes ssDNA overhangs that can anneal to their ssDNA complement;
(ii) DNA polymerase activity, that fills gaps in the annealed products, and
(iii) DNA ligase activity, that covalently seals the resulting nicks in the assembly.
示意图：
组装特点：
需要给每个片段设计15-20 bp的同源片段。
试剂盒：
选用了NEB的Gibson Assembly
Master Mix（E2611L）。这些资料也是从NEB网站上转载的。详细的内容大家可以参考NEB网站。
网址（总体介绍）：
https://international.neb.com/tools-and-resources/feature-articles/gibson-assembly-building-a-synthetic-biology-toolset
网址（试剂盒）：
https://international.neb.com/products/e2611-gibson-assembly-master-mix#Product%20Information
工作手册：
https://international.neb.com/-/media/nebus/files/manuals/manuale2611.pdf?la=en&rev=9db62577a41b4cfda071e21864a6763e&hash=09806AE9A3D8C9C7FDA0A58030AA9F7F9FD1A56F
试剂盒手册：
https://international.neb.com/-/media/nebus/files/manuals/manuale5510.pdf?la=en&rev=c677dffb8d694d948da64c31b3d85e30&hash=7294EED0EB8F388C9100AA2C6819C0D865C3605D
详细的内容大家可以去仔细看一下原文。
具体的protocol：
1、计算体系中含有的所有DNA的量：
NEB recommends a total of 0.02–0.5 pmols of DNAfragments when 1 or 2 fragments are being assembled into a vector and 0.2–1.0pmoles of DNA fragments when 4–6 fragments are being assembled. Efficiency ofassembly decreases as the number or length of fragments increases. To calculatethe number of pmols of each fragment for optimal assembly, based on fragment lengthand weight, we recommend the following formula:
pmols = (weight in ng) x 1,000 / (base pairs x 650daltons)
50 ng of 5000 bp dsDNA is about 0.015 pmols.
50 ng of 500 bp dsDNA is about 0.15 pmols.
The mass of each fragment can be measured using theNanoDrop instrument, absorbance at 260 nm or estimated from agarose gelelectrophoresis followed by ethidium bromide staining.
2、反应体系：
1.
* Optimized cloning efficiency is 50–100 ng ofvectors with 2–3 fold of excess inserts.
Use 5 times more of inserts if size is less than200 bps. Total volume of unpurified PCR fragments in Gibson Assembly reactionshould not exceed 20%.
** Control reagents are provided for 5 experiments.
*** If greater numbers of fragments are assembled,additional Gibson Assembly Master Mix may be required.
2. Incubate samples in a thermocycler at 50°C for15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6fragments are being assembled. Following incubation, store samples on ice or at–20°C for subsequent transformation.
总而言之，50-100 ng的载体，载体2-3倍的片段浓度，如果片段比较小，如小于200 bp，需要调整片段浓度到载体的5倍。连接可以比1小时要长，但是不要连接过夜。
3、转化步骤：
1. Thaw chemically competent cells on ice.
2. Transfer 50 μl of competent cells to a 1.5 mlmicrocentrifuge tube (if necessary).
3. If the chemically competent cells are from NewEngland Biolabs, add 2 µl of assembled product to NEB competent cells and go tostep 4 directly. If competent cells are purchased from other manufacture, dilute assembled products 4-fold with H2O prior transformation. This can be achieved by mixing 5 µl of assembled products with 15 µl of H2O. Add 2 μl of the diluted assembled product to competent cells.
如果使用的不是NEB的菌菌，建议产物稀释4倍，加2 µl的量进去转化。
4. Mix gently by pipetting up and down or flicking the tube 4–5 times. Do not vortex. Place the mixture on ice for 30 minutes. Do not mix.
5. Heat shock at 42°C for 30 seconds.* Do not mix.
6. Transfer tubes on ice for 2 minutes.
7. Add 950 μl of room temperature SOC media* to tubes.
8. Place the tube at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
9. Warm selection plates to 37°C.
10. Spread 100 μl of the cells onto the plates with appropriate antibiotics. Use
Amp plates for positive control sample.
1 ml体系使菌菌恢复，再取十分之一的菌菌涂板。
11. Incubate plates overnight at 37°C.
* Please note: Follow the manufacturer's protocols for the duration and temperature of the heat shock step, as well as the optimal medium for recovery. Typically, transformation of our positive control assembly product will yield more than 100 colonies on an Amp plate with greater than 80%colonies containing inserts.
SOC media的成分表（可能可以自己配制）：
1X SOC Outgrowth Medium:
2% Vegetable Peptone
0.5% Yeast Extract
10 mM NaCl
2.5 mM KCl
10 mM MgCl2
10 mM MgSO4
20 mM Glucose
写在最后：
我还没有实践这个方法，可能实践以后会有其他问题或者注意事项~希望实验顺利~