IGEM:Hong Kong HKUST/2009/Notebook/Effects of vector to insert DNA ratio on ligation efficiency/Entry Base
- Chen Rui, Raymon
- Cheng Ka Yan
- Tsang Chor Shan, Candy
- Cheng Kar On
This experiment is about the ligation of BBa_K823005 and BBa_K516030 and finding the optimal ratio of the vector and insert. The biobrick at the end is made by a vector and an insert. In this case, part of BBa_K516030 is cut and inserted to BBa_K823005. The aim of this experiment is to investigate the effects of vector to insert DNA ratio on ligation efficiency, so sets with different ratio are compared. Finally, the finding shows that the optimum molar vector to insert DNA ratio of four groups is 1:3.
Ligation which is part of the cloning methods can join two linear DNA fragments together by forming covalent bonds with the help of ligase. By using this method, the DNA in cells can be altered.
This experiment is to ligate BBa_K823005 and BBa_K516030. Before ligation, a series of cloning processes are needed. Four different ratios of the vector and insert (1:0.5, 1:1, 1:3, and 1:10) are used for ligation. Transformation is applied again. At the end, the number of red colonies which represents successful ligation is counted and its percentage is then calculated. Cells in red colonies possess desired DNA while cells in the white may lack insert or being plasmid-free.
In this experiment, BBa_K823005 and BBa_K516030, which follow the RFC10 standard, are used because BBa_K823005 has a promoter while BBa_K516030 has ribosome binding site, reporter gene and double terminator. When they are put together, the whole DNA is functional and protein can be produced. In this experiment, BBa_K823005 and BBa_K516030 are the vector and insert respectively.
Therefore, specific enzymes could be used to cut the site XbaI and PstI of BBa_K516030 and the site of SpeI and PstI of BBa_K823005. Ligase is then used to insert the fragment of BBa_K516030 into BBa_K823005. A strong covalent bond called “scar” is formed between the X and S site.
Finding the optimal ratio is helpful for future experiments, for instance, it would be more efficient if someone needs to put two biobricks together. If someone needs to use this biobrick for his/her other research topics, using the optimal ratio for ligation can reduce the experimental errors.
Methods and Materials
Two plasmids, pSB1C3-BBa_K823005 and pSB1C3-BBa_K516030, were transformed into Escherichia coli (E. coli) DH10B strain respectively. To get enough plasmids, they were inoculated in Lysogeny Broth with chloramphenicol at 37 °C overnight. Plasmids were extracted out in the next day according to the Mini Plus™ Plasmid DNA Extraction System protocol. The concentration of the extracted DNA was measured by NanoDrop spectrophotometer. Next, the digestion of plasmids was proceeded to obtain target vector and insert. Four experimental groups together with four control groups without ligase were set. For digestion recipe, pSB1C3-BBa_K823005 groups were digested by PstI-HF® and SpeI-HF® while pSB1C3-BBa_K516030 groups were digested by PstI-HF® and XbaI. The volumes of all groups were brought to 20 μl with ddH2O. The length of expected vector is 2087 base pairs and expected insert is 897 base pairs. Gel extraction was then followed by gel purification which was done according to the Favorgen FavorPrep™ GEL/PCR Purification Mini Kit protocol. The linear DNA fragments were collected. The concentration of the digested DNA was assessed in order to calculate the volume of reagents.
|1:0.5||1:0.5 (c)||1:1||1:1 (c)||1:3||1:3 (c)||1:10||1:10 (c)|
|vector 150ng (μl)||6.58||4.24||6.58||4.24||6.58||6.58||6.58||6.58|
|ligase buffer (μl)||1||1||1||1||1||1||1||1|
|T4 DNA ligase (μl)||1||0||1||0||1||0||1||0|
Due to different DNA concentration after ligation, the total volume of each group various. The reaction mixtures were put at room temperature for 1.5 hours. These recombinant plasmids were transformed into E. coli DH10B strain and incubated in chloramphenicol agar plates overnight. The number of red colonies and the total number of colonies could be counted next day.
For each group, the numbers of red colonies were divided by the total number of total colonies was the ligation of efficiency. The result was shown in the table below.
|Red colonies||White colonies||Total Colonies||Efficiency (%)|
As shown in table 2, the 1:3 ratio group had the largest number of colonies and the highest ligation efficiency among the four. After some comparison, it indicated that the ligation efficiency tended to increase with the increase percentage of inserts. At a specific molar ratio, the efficiency reached the optimal value and then decreased. A conclusion could be drawn that the optimum molar ratio of vector BBa_K823005 to insert BBa_K516030 is 1:3 under this experimental conditions.
It is shown that the optimum molar ratio is 1:3 as it has the highest number of colonies. In addition of that, there is an increase of successful colony with a higher fraction of inserts. However, the efficiency of ligation turns out to decrease as the 1:10 group has fewer colonies than that of 1:3 group. Therefore, a simple linear relationship does not apply to the molar ratio of vector to insert and the successful ligation efficiency. It suggests that the ratio should be neither too high or too low and there exists an optimum value for the ratio. It has been reported by James (n.d.) that if the molar ratio of vector to insert is too high, plasmids without insert and polymeric plasmids may be produced as the cohesive ends of DNA of vector are the same; if the ratio is too low, one vector may tend to ligate with two or more inserts. The result of ours corresponds well to James’. In the end, both colony PCR and restriction check were carried and it turned out most of these colonies were correct.
The result implies an optimum molar ratio of vector pSB1C3-BBa_K823005 to insert BBa_K516030 for getting an efficient ligation and subsequent transformation is 1:3. And it provides a reference to those whose experimental material is similar. For a more accurate optimization, it has been proposed that more sets of parallel experiment between 1:3 and 1:10 are needed. In addition of that, since T4 DNA ligase is very sensitive to shear and ligase buffer contains unstable ATP, the experimental operations contribute a lot to the final yields. At room temperature, the reaction rate is fast but the T4 DNA ligase and ATP are very unstable; on the contrary, at 16 °C, the reaction rate is much lower but the T4 DNA ligase and ATP are more stable. In view of this, for those who have enough time, culturing bacteria at 16 °C in an incubator for a longer time is a good choice to get higher efficiency; but more often, the efficiency may be improved by additional T4 DNA ligase and ATP. In subsequent experiments, more factors such as the size of vector and insert and the duration of ligation should also be considered.
Based on the result above, it is concluded that the molar ratio of vector to insert has a significant effect on the outcome of ligation and the subsequent transformation step. For this experiment, the optimum molar vector to insert DNA ratio of four groups is 1:3.
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