The Use Of Chromoproteins For Positive Identification Of Gene Expression

Abstract

DNA cloning and expression vectors are the modern-day mainstays for molecular biology studies. In Spite Of advances in synthetic biology and modern techniques, researchers around the world still uses the standard way of DNA cloning, using plasmids and reporter proteins for selecting possible recombinant DNA. The coloured properties of blue coral chromoproteins (AmilCP) and red fluorescent protein (Rfp), from Acropora millepora and Discosoma striata respectively, makes them a suitable reporter protein. These genetically encoded reporter proteins turned out to be a distinct tool that assisted direct visualization of structures and processes in living cells and organisms. We tried to insert these chromoproteins and fluorescent proteins into the plasmid pTTQ18 and transforming them into the bacteria Escherichia coli (E. coli). The quantification of the coloured and/or visible gene expression was done to check for the success in the method of expression.

Introduction

Plasmids are well known for their use in synthetic or molecular biology lab for their extensive use as expression vectors (Sriram Padmanabhan, Naganath Mandi and Sampali Banerjee, 2011). From the time when the first recombinant molecule was generated by Jackson and colleagues (Jackson, Symons and Berg, 1972), advances in the understandings of how the fundamental process in living organisms’ function were able to be uncovered using the recombinant DNA. Plasmids with more complex nature allows incorporation of broader hosts and wider applications as well (Jackson, Symons and Berg, 1972) (Baek et al., 2014). The coral evolved through several autocatalytic reactions to synthesize coloured or fluorescent compounds known as the chromophore (D’Angelo et al., 2008). Even after with all the advancement in the development of the plasmid there could be ways to further improve the plasmid cloning by improving the identification of the positive clones (Tafoya-Ramírez et al., 2018).

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The aim of this study was to incorporate chromoproteins successfully and to positively identify clones by naked eye. The process of molecular cloning or forming a recombinant DNA requires the presence of two important units a vector and the gene of interest (GOI). The vector and the GOI is usually cut in the desired section with the help of an enzyme restriction endonucleases. Any vector and DNA that is cut by the same restriction enzyme will join by their respective base pairs according to the compatibility of their respective ends. The joining of the vector with the required DNA is brought about by the action of DNA ligase by forming phosphodiesterase bonds. The expected recombinant DNA is then amplified in the PCR and then transformed into the bacteria. The pTTQ18 vectors has a polylinker region on the lacZ location. We tried to insert a foreign gene, the chromoprotein, into this region for recombinant purpose and expression of the desired reporter protein.

Materials and Method

The reporter protein was obtained from the International Genetically Engineered Machine (iGEM). The vector pTTQ18 and the reporter protein, 10 µL each, were taken into a separate microcentrifuge tube for restriction digest (Loenen et al., 2013). The restriction endonucleases smaI and kpnI, 1μL each, were added in both the tube containing the DNA with 6 μL of sterile water and incubated at 370C for 1 hour. The resulted product was gel purified in 1% agarose gel with 4 μL loading buffer and 20 μL of each of the digested DNA. The gel was run for 30 minutes at 100 V and viewed using GelRED under UV. The ligation of the purified-digested DNA was brought about using 25 μL of the DNA ligase enzyme and left for incubation at 150C overnight. To check the correct insertion of the reporter protein sanger sequencing by Capillary Electrophoresis method was used (Sanger, Nicklen and Coulson, 1977) (Smith, 1991).

The recombinant DNA was then merged into the E. coli JM101 cells by the process of chemical transformation (Ren et al., 2019) (Rahimzadeh and Sadeghizadeh, 2016). We used 5 μL of the ligation mix with 50 μL of the E. coli cells and used 950 μL of L-broth, which was incubated for 30 minutes at 370C. 50 μL of the sample broth was plated in nutrient agar ampicillin plate following proper plating methods (Sanders, 2012).

Discussion

A recombinant DNA was successfully constructed using the ptac promoter and Red fluorescent protein of Discosoma striata. These DNA once transformed into the E. coli makes multiple copies of themselves. The expression of the Rfp in the bacterial colonies were visible to the naked eye and did not require the use of any treatment of chemical or special instruments. The colonies formed were counted and found to be 67 white and 25 red colonies, meaning 25 of the cells was successfully transformed with the recombinant plasmid. Some larger colonies were observed which could be due to improper streaking over the agar medium.

The sequence from the chromatogram was used for a BLAST hit showing a 100% match of the sequence with the A. millepora. The alignment (Figure-5) sequence with vertical lines indicate the locations of the identical residues. The results indicate that the cloning plasmid formed using the PTTQ18 backbone was stable and we have successfully cloned the reporter protein into the pTTQ18 vector. This colour phenotype of the transformants make the system compliant and easily detectable.

The plasmids depending on the chromoprotein, AmilCP, as the reporter protein are being considered in the Golden-Gate method as well (Andreou and Nakayama, 2018). However, the usual method of cloning is still followed all over the world that includes vectors which are user friendly and cheaper selection markers. This study provides a reasonable way of detecting positive clones without the use of any special instruments. These particular markers can be a promising tool for mutant selection alongside other reliable positive cloning selection with reporter proteins that are readily detected with the naked eye (Choe, 2005). The strategy presented here can reduce cloning cost meaning a cheaper method.

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