Gibson Assembly
這個方法是在一個反應體系里面，一步合成多個片段的基因組裝技術。
具體的原理是：
1.? ? 核酸外切酶從5’端移除單個核苷酸，同源片段通過anneal（退火）搭在一起；
2.? ? DNA聚合酶以某一鏈為模板，補充缺失的片段；
3.? ? ?DNA連接酶形成磷酸二酯鍵，最后產生完整、沒有連接縫隙的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.
示意圖：
組裝特點：
需要給每個片段設計15-20 bp的同源片段。
試劑盒：
選用了NEB的Gibson Assembly
Master Mix（E2611L）。這些資料也是從NEB網站上轉載的。詳細的內容大家可以參考NEB網站。
網址（總體介紹）：
網址（試劑盒）：
https://international.neb.com/products/e2611-gibson-assembly-master-mix#Product%20Information
工作手冊：
試劑盒手冊：
詳細的內容大家可以去仔細看一下原文。
具體的protocol：
1、計算體系中含有的所有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、反應體系：
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.
總而言之，50-100 ng的載體，載體2-3倍的片段濃度，如果片段比較小，如小于200 bp，需要調整片段濃度到載體的5倍。連接可以比1小時要長，但是不要連接過夜。
3、轉化步驟：
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，建議產物稀釋4倍，加2 μl的量進去轉化。
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體系使菌菌恢復，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
寫在最后：
我還沒有實踐這個方法，可能實踐以后會有其他問題或者注意事項~希望實驗順利~
