IGEM:Hong Kong HKUST/Investigations/The Effect of the Vector to Insert DNA Ratio on the Efficiency of Ligation

Authors CHEUNG Ming Fung Alice Wang Qingyang Amanda LIUSNANDO Abstract It is believed that ligation process is highly dependent on the ratio of vector to insert DNA. We had performed experiment to investigate 4 vector to insert molar ratios, including 1:1, 1:3, 1:10, 1:30. We used pSB1C3-BBa_K880005 as the backbone, BBa_J04650 as the insert, Escherichia coli. (DH10B) to hold the ligation product, and red fluorescence protein (RFP) to show the result. The result indicated that 1:10 ratio yields the most desired efficiency. Introduction DNA ligation is the process of forming a phosphodiester bond between the 3' hydroxyl of one nucleotide and the 5' phosphate of another to join together two DNA molecule ends. Usually, the process ligases the DNA of interest which is called the insert DNA with a backbone vector. However, either excess insert DNA or vector may cause low efficiency of desired ligation. If the vector to insert ratio is too high then 'empty' mono and polymeric plasmids will be generated. If the ratio is too low then excess insert fragments tend to ligate together producing non-productive ligation results. In other words, there should be enough insert DNA to form productive ligation product but not too much to cause oligomers. Therefore, we would like to investigate the optimum vector to insert molar ratio by using 1:1, 1:3, 1:10 and 1:30. Methods and Materials BBa_pSB1C3-J04650 and pSB1C3-BBa_K880005 plasmids were transformed into E. coli which were later inoculated into Lysogeny Broth for overnight incubation with chloramphenicol antibiotic. Plasmids were then extracted with GTpure™ Plasmid miniprep purification kit. The concentration and quality of purified DNA sample was assessed by NanoDrop 2000 spectrophotometer. Approximately 3μg of pSB1C3-J04650 was digested with 10 units of XbaI-HF® and PstI, 1μg of pSB1C3-K880005 was digested with 3 units of PstI and SpeI, in 1X NEB Buffer. We extracted the digestion product by doing electrophoresis and purified the extraction by FAVOGEN GEL/PCR purification kit. The concentration of purified DNA samples were assessed by NanoDrop 2000 spectrophotometer. Then we carried out our experiments in 6 different sets, involving 4 sets of different insert to vector ratio and 2 negative controls. The ligation recipe is listed as follow: Table 1. Ligation recipe for 4 sets of experiments and 2 negative control groups. We incubate the prepared mixture at room temperature for 30 minutes then transformed the product into DH10B E.coli for overnight incubation at 37℃ in the chloramphenicol selection plate and observe the presence of red colony the next day. Results and Interpretations Figure 1. Plate image after overnight incubation at 37℃ of the ligation product. The first row from left to right: negative control without insert DNA, negative control without vector; the second row from left to right: 1:1 ratio set, 1:3 ratio set; the third row from left to right: 1:10 ratio set, 1:30 ratio set. After counting the total colonies, the red colonies and the white colonies, a table below can be filled with the counting result. Table 1. The amount of colonies of different kinds after overnight incubation at 37℃ and the according ligation efficiency. As shown in Table1.all negative control groups show no or little colonies. The red colony formed on negative control could be a contamination. The 1:30 ratio group also showed no present of colonies. The reason behind may be too many insert oligomers have been ligated which is linear and could not be ligated into a circular plasmid. The other groups all showed colonies on the plates thus a graph of efficiency against ratio can be shown below: Figure 2. The effect of insert to vector ratio on the efficiency of ligation. By calculating the red colonies over total colonies ratio we can have the result as the ligation efficiency which is indicated in the graph as the vertical coordinate, take the molar ratio of vector to insert DNA we have the horizontal coordinate thus the graph above may show the effect of vector to insert DNA ratio on the efficiency of ligation. From Figure2.we can see that the efficiency of ligation increases as the vector to insert DNA molar ratio changes from 1 to 3 and reaches maximum at 10 and then reaches the lowest when the molar ratio is 30 according to our experiment sets. It indicates that 1:3 or 1:10 molar ratio yields the optimum ligation efficiency. The result met our expectation to yield the optimum ratio in the middle of the range and specifically, we can come to the conclusion that when the molar ratio of pSB1C3-K880005 to J04650 is around 10, the ligation process yields the optimum efficiency. Discussion We adjusted the amount of DNA which will be used in a ligation based on the length of the DNA and checked it by using ligation calculator. It is shown that the optimum molar ratio of vector to insert is 1:10 as it yielded the highest number of successful colony has been ligated. It also can be seen that there is an increasing trend of successful ligation colony with higher molar ratio of the vector to insert from 1:1 to 1:10. However, there is an absence of colony when the ratio of the vector to insert is 1:30. Therefore, our results suggest that the ligation process will not be optimized when the ratio of the vector to insert is too high or too low and is optimum at the ratio of 1:10. These findings are in accordance to the theory that low ligation efficiency of desired clone can be caused by excess insert. According to Judelson Laboratory at the University of California, this absence of colony in high vector to insert molar ratio may be caused by the inclination of vector to ligate to 2 or more insert and the tendency to form dimer and trimmers. It is also suggested that the optimum ratio can be achieved when there is enough concentration of termini to favor intermolecular ligation but can not be so high and from our results, it implies that the ratio 1:10 is this optimum ratio. Some contamination may have occurred during the experiments which led to the appearance of colony in the control. As T4 DNA Ligase is very sensitive, our results may also have been affected by incautious mixing or vortex of it. The result that we have obtained can be used as a standard for achieving the most effective ligation process in the upcoming use of pSB1C3-BBa_K880005 and BBa_J04650. More improvement can be done by using various range of different ratios including 1:6 , 1:15 or 1:20 to see the possibilities of higher yields or lower yields that may be arisen. Another step that can be done to improve our result is to purify the ligation by using phenol and chloroform which can be followed by ethanol precipitation overnight as this method may boost the transformation efficiency, as reported by Wellcome Trust Sanger Institute, Cambridge. There may be another factors that can affect the ligation, such as the length of the molecule, the DNA size, the conditions of whether the ends are cohesive or blunt and therefore different vector or insert been used may emanate different results. Conclusion Different molar ratio of vector to insert has shown different result in the efficacy of ligation process. Based on the results obtained, it can be concluded that The efficiency of ligation process is affected by the molar ratio of vector to insert and the optimum molar ratio of vector to insert is 1:10.