IGEM:Hong Kong HKUST/Investigations/Effect of Dephosphorylation on Reduction of Background Ligation

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Authors

Abstract

This experiment was conducted to see the effect of removing the phosphate group in both 5’ end of linear DNA backbone on background ligation, which in our case is the self-ligation of the backbone. Self-ligation will increase the amount of vector containing no insert DNA. Therefore, by removing the phosphate from 5’ end can inhibit the formation of phosphodiester bond that is required for DNA ligation to proceed, and inhibit self-ligation. The result of reduced background ligation, with increasing percentage of RFP producing colony was expected. Unfortunately, the colony ratio of normal backbone with RFP to dephosphorylated backbone with RFP was way higher than expectation, and colony counting and a valid comparison were impossible to be done, due to the extremely high amount of normal backbone with RFP colonies, thus our result is inconclusive.

Introduction

During the ligation of foreign DNA fragment and DNA vector, self-ligation is one of the most influential sources of background ligation that induces a high background of transformants, which contain recircularized vector DNA without inserts.

The vector contains a 5’ phosphate group and also a 3’ hydroxyl group at its terminals. During the process of ligation, phosphodiester bonds form between the 5’ phosphate group and 3’ hydroxyl group under the catalysis of DNA ligase. As a result, the removal of 5’ phosphate groups in both ends of the linear DNA with phosphatase can avoid self-ligation. Nonetheless, ligation of foreign DNA fragments with 5’ phosphate group to the dephosphorylated backbone can still be done efficiently as phosphodiester bonds can be formed between the 5’-end phospate group of the foreign DNA and the 3’-end hydroxl group of the vector. Thus, the number of transformed bacteria, which do not contain the correct insert, can be reduced.

In our experiment, we examined the effect of dephosphorylation of vector DNA by transforming gene containing normal vector and dephosphorylated vector respectively, together with red fluorescent protein coding device (BBa_J04450) as reporter into the Escherichia coli DH10B strain, followed by comparing the amount of RFP expressing colonies of the two setups. Note that the colonies, which do not show RFP expression, contain self-ligated vector, while those showing RFP expression do not.

Methods and Material

Plasmid Construction To investigate the effect of dephosphorylated vector in background ligation, a gene encodes for monomeric red fluorescent protein (mRFP) were constructed with the normal and dephosphorylated vector respectively and were transformed into E. coli DH10B strain. Cells were then investigated after being streaked onto agar plates .

Set-ups
Insert Vector
BBa_J04450 pSB1AK3
BBa_J04450 pSB1AK3 (Dephosphorylated)
pSB1AK3
pSB1AK3 (Dephosphorylated)











The two parts were assembled using the RFC10 standard, with pSB1AK3 as the backbone. Negative controls were constructed using solely the vectors pSB1AK3 and dephosphorylated pSB1AK3 respectively. All constructed genes were transformed into E. coli DH10B strain.

Dephosphorylation of pSB1AK3

1. Add 1 ul of Antarctic Phophatase and Antarctic Phophatase buffer (1x in the final concentration) to dephosphorylate pSB1AK3
2. Incubated for 1 hour at 37°C
3. Heat inactivated at 65 for 5 minutes (using thermocycler)

Ligation

1. 20 ng of vector DNA (Vector to Insert ratio of 1:2) is used
2. 0.5 ul T4 Ligase and T4 Ligase buffer (1x in the final concentration) added
3. Incubated at room temperature for 2 hours.

Transformation

1. 13.5 of ligated DNA is used 2. incubated in ice bath for 15 mins.
3. 5 mins heat-shock at 37°C.
4. 2 mins in ice bath
5. 1mL LB used and incubated in 37°C for 1 hour.
6. Spread on LB plate with kanamycin.

Result and Interpretation

Colony normal dephosphorylated vector.jpg
Figure 1. Effect of Vector Dephosphorylation. One set used normal pSB1AK3 vector to ligate with BBa_J04450. Another set used dephosphorylated pSB1AK3 vector to ligate with BBa_J04450.

As qualitatively observed, the amount of colonies which shows RFP expression in all 3 normal backbone with RFP setups are extremely high compared to the that of the dephosphorylated backbone with RFP setups -- Edward’s dephosphorylated backbone with RFP setup shows only a few colonies and with few colonies giving RFP expression; while the other 2 dephosphorylated backbone with RFP setups show no colonies. Also, only a very few number of colonies were shown in the dephosphorylated backbone controls. Some red colonies were also observed in our control for normal backbone, which maybe due to contamination.

Discussion

It is shown that the dephosphorylated backbone with mRFP setups shows no or just a few conlonies while the normal backbone with RFP setup shows a lot more colonies. The number of colonies in the dephosphorylated backbone with RFP setup is unexpectedly low, it is believed that the abnormality is due to the certain errors in process of dephosphorylation.

It is logical to speculate the reason of obtaining the aforementioned observation is that most of the bacteria in the plate do not contain or merely carry incomplete dephosphorylated backbone with RFP plasmid, and hence were killed by kanamycin. While it is still in interest to determine the reason(s) triggering the situation that the transformant doesn't contain or only contains incomplete DNA.

Firstly, the situation is possibly due to the instability of the dephosphorylated backbone. The backbone itself might not be as stable as the normal backbone, and thus having a higher chance to be lost during the incubation. The second is reason is that there might still be trace amount of phosphatase. Heat might not be 100% working to inactivate the phosphatase. The phosphatase might also dephosphorylate the insert, and during ligation, phosphodiester bond cannot be formed, thus less vector is ligated to the mRFP generator.

Conclusion

Our investigation is inconclusive, it is due to the failure in the dephosphorylated-backbone setup. As a result, we cannot count the number of colonies in the setup, and hence compare the number of RFP expressing colonies between the normal-backbone setup and the dephosphorylated one.

Reference

Dephosphorylation. (n.d.). Retrieved June 8, 2015, from https://www.neb.com/applications/cloning-and-synthetic-biology/dna-end-modification/dephosphorylation

Aitken, A. (n.d.). Dephosphorylation of DNA for Ligation. Retrieved October 11, 2012, from http://www.nhm.ac.uk/resources-rx/files/dephosphorylation-of-dna-for-ligation_aug12-118533.pdf