IGEM:HKUST iGEM 2016/2009/Notebook/Effect of Dephosphorylation on Reduction of Background Ligation/Entry Base
This experiment aimed to study the effect of dephosphorylation on reduction of background ligation. By cutting out the desired vectors and inserts, followed by ligation, the required plasmids pSB1C3-BBa_J04450 were expected to be formed. Dephosphorylation was conducted on one set of vectors before ligation, and ligations with no insert were also done as control experiments. After transforming the plasmids into DH10B Escherichia coli (E. coli), the phenotype of the plasmids, red fluorescence, was observed. At last, by comparing the percentage of red colonies formed on agar plates, the effect of dephosphorylation on reduction of background ligation was expected to be found. However, due to unexpected absence of colonies in dephosphorylated vectors plates, quantitative analysis of percentage of red colonies was impossible. This experiment is thus inconclusive.
Dephosphorylation is a common step in recombinant DNA technology that facilitates ligation by prohibiting self-ligation of the digested DNA. Self-ligation is considered as one of the main sources of background ligation, in which it may hinder the successful ligation of the desired insert into the DNA vector.
As phosphodiester bonds are formed between the 5’ phosphate group and 3’ hydroxyl group by DNA ligase during ligation, the removal of the 5' phosphate group of the linearized DNA by phosphatase could reduce the occurrence of intramolecular recircularization and hence reducing background ligation during subsequent transformation. After dephosphorylation, phosphodiester bonds could be formed more effectively between dephosphorylated vectors and inserts.
The objective of the experiment was to investigate the effect of dephosphorylation on reduction of background ligation. In the experiment, both normal vectors and dephosphorylated vectors were studied. Normal vectors and dephosphorylated vectors were allowed to form colonies, either with or without inserts, after transformation. With insert encoding the gene of red fluorescent protein (BBa_J04450) together with vector (pSB1C3), the constructed plasmid (pSB1C3-BBa_J04450) was transformed into the Escherichia coli strain DH10B, the percentage of colonies with RFP expression were compared among normal vectors and dephosphorylated vectors’ set-up. Note that colonies showing RFP expression are without self-ligation and vice versa, the effect of dephosphorylation on reduction of background ligation could be examined.
Methods and Material
In order to test the effect of dephosphorylation on reduction of background ligation, inserts encoding the gene of red fluorescent protein (BBa_J04450), were constructed with either normal backbones or dephosphorylated backbones (pSB1C3). Negative controls were done by using exclusively normal backbones or dephosphorylated backbones respectively.
Dephosphorylation of pSB1C3
1. 1/10 of total reaction volume of 10X Antarctic Phosphatase Reaction Buffer was added to the purified DNA mixture.
2. 1 ul (5 units) of Antarctic Phosphatase was added and mixed.
3. Reaction mixture was incubated at 37°C for 15 minutes
4. Microcentrifuge tubes were incubated at 70°C for 5 minutes to heat inactivate the enzyme.
1. Reaction mixture was set up in microcentrifuge tubes on ice.
2. Reaction mixture was mixed gently by pipetting up and down briefly.
3. Microcentrifuge tubes were incubated at room temperature for 10 minutes.
4. Microcentrifuge tubes were incubated at 65°C for 10 minutes to heat inactivate the enzyme.
5. All microcentrifuge tubes were put on ice and 1-5 ul of reaction was transformed into 50 ul E. coli.
6. All microcentrifuge tubes were incubated at 37°C for 1 hour.
1. Competent cells (DH10B E. coli) were thawed on ice for around 25 minutes.
2. Agar plates were warmed by placing them in 37°C incubator.
3. 1-5 ul of DNA was mixed with 20-50 ul of competent cells in a microcentrifuge tube, bottom of tube was flicked with finger a few times to ensure gentle mixing.
4. Competent cell/ DNA mixture was put on ice for 20 minutes.
5. All microcentrifuge tubes were heat-shocked by placing them into a 42°C water bath for 1.5 minutes.
6. Tubes were put back on ice for 2 minutes.
7. 450 ul of LB (without antibiotic) was added into each tube and grown in 37°C shaking incubator for 45 minutes.
8. All the tubes were centrifuged at 7000 rpm for 3 minutes.
9. 250 ul of each tube of transformant was transferred onto a LB agar plate with appropriate antibiotic (KAN and CHL).
10. All the plates were spread and incubated at 37°C overnight.
Result and Interpretation
By comparing the percentage of coloured colonies, effect of dephosphorylation on reduction of background ligation could be examined .
For sample plates with inserts added, no red colony but a few white colonies were found on dephosphorylated vectors plates. Higher percentage of red colonies were present on normal vectors plates.
For control plates with no inserts added, no colony was found on dephosphorylated vectors plates. Many white colonies were found on normal vectors plates.
For sample plates with inserts added, no red colony but only a few white colonies were found on dephosphorylated vectors plates. Higher percentage of red colonies were present on normal vectors plates. This might be caused by errors in the step of dephosphorylation.
Heat inactivation was needed after dephosphorylation of the pSB1C3 vectors. However, the time and temperature used to inactivate the restriction enzyme might not be sufficient. Thus, some phosphatase might still exist in the sample mixture, which might further dephosphorylate the inserts. As a result, ligations would not be successful, phosphodiester bonds were not formed between the end of inserts and plasmids and circular dephosphorylated vectors-inserts plasmids were not formed. Linearized vectors and inserts could not be transformed into E. coli and thus, red colonies were not present in the samples.
This experiment was inconclusive due to the unexpected results found on dephosphorylated vectors samples, quantitative analysis of percentage of red colonies was impossible. More red colonies were found on normal vector plates rather than dephosphorylated vector plates, which is opposite to our hypothesis. The objective of this experiment, which is to prove dephosphorylation can reduce background ligation, was not achieved.
Dephosphorylation. (n.d.). Retrieved Jan 27, 2016, from https://www.neb.com/applications/cloning-and-synthetic-biology/dna-end-modification/dephosphorylation#tabselect1
T4 DNA Ligase. (n.d.). Retrieved Jan 27, 2016, from https://www.neb.com/products/m0202-t4-dna-ligase