Escherichia coli (BL-21 strain) plasmids pMICO and pGLO were isolated and identified using 0.8 % agarose gel electrophoresis.

Aims and Backgrounds:Human Computer Interaction

Recombinant DNA technology is especially attractive to humans because it could lead to the development of vaccines against serious diseases by reengineering cells to produce foreign proteins. (Jonas, 2021). Foreign DNA can be inserted into a bacterial host such as Escherichia coli (E. coli) BL21 by cloning it into a plasmid known as a vector (DE3). A plasmid is a circular DNA molecule that replicates on its own and is found in bacteria and other microscopic organisms. It contains a small number of genes associated with antibiotic resistance and can be passed between cells. (Clark et al., 2019)

The aim of this experiment was to identify and isolate two distinct E. coli cultures (pMICO and pGLO) that could be used for studying Plasmodium falciparum using fluorescently labelled malarial proteins. P. falciparum malaria is a disease that causes 90% of deaths worldwide and is a public health concern. (Zekar & Sharman, 2022). The disease will be studied using pMICO plasmid (which encodes a protein) and pGLO plasmid (which encodes a green fluorescent protein (GFP)). In this E. coli host strain, a recombinant plasmid (vector + foreign DNA) allows for a variety of genetic manipulations including directed mutations, recombinant protein expression, and fluorescent tagging. (Nelson & Cox, 2013). In addition, the malaria parasite has different codon preferences during translation in E. coli, resulting in low protein expression levels. This was largely resolved by introducing a tRNA-encoding plasmid (RIG plasmid) construct that recognised these rare codons in E. coli. However, this system has limitations when it comes to studying proteins that are toxic to E. coli, such as pMICO. This system was created to express proteins cloned from P. falciparum that would otherwise be toxic to host E. coli. (Cinquin et al., 2001)

This was achieved by isolating the plasmids pMICO and pGLO from their bacterial hosts. The samples were digested by single and double digesting the plasmids and then transferred to an 0.8 % agarose gel for analysis. However, the E. coli (BL21) strain must be confirmed to contain the plasmid pMICO before further experiments can be conducted. In this experiment, Luria-Bertani broth (LB) was used for cultivating this bacterial culture (see appendix 1), with 50 μl of chloramphenicol stock solution added (see appendix 2). In both pMICO and pGLO plasmids, restriction enzymes recognize a single recognition site, usually 4-6 base pairs; hence, double digestion is necessary. In pMICO, there are three restriction enzymes: Not1 and Sal1, a double digest of Not1 and Sal1, and an undigested plasmid (control); in pGLO, there is only one double digest, Pst1, and an undigested plasmid (control). Likewise, both plasmids required the same buffer D and were incubated at 37 °C. Furthermore, Not1 and Sal1 provide double digestion, making them the most effective restriction enzymes. The NanoDrop^TM One has also been used for determining the DNA purity and concentration of pMICO and pGLO plasmids (see appendix 4). Additionally, the digested and undigested plasmids of pMICO and pGLO will be analysed on an 0.8% agarose gel for approximately 68 minutes to determine if the observed bands match expected fragments.

Results and Discussion:

Table 1: A sample of both pMICO and pGLO plasmids with undigested and digested plasmids. A pMCO plasmid contains three restriction enzymes: two single-digested Not1 and Sal1, one double-digested Not1 and Sal1, and an undigested pMICO plasmid (control), while a pGLO plasmid contains double-digested Pst1 and an undigested plasmid (control).

Figure 1: GelDoc BioRad 0.8% agarose gel electrophoresis system was used to capture this image. Lane 1 shows a 1kb DNA ladder from Promega; lane 2 is an undigested plasmid of pMICO (control); lane 3 shows Sal1 which is single digested; lane 4 shows Not1 that has been single digested; lane 5 shows NotI and SalI double digested from pMICO plasmid; lane 6 is the undigested plasmid (control) of pGLO and in lane 7 is pGLO double digested, Pst1.

Lane 1 displays a 1kB DNA ladder from Promega. The undigested plasmid (control) of pMICO (lane 2) has a blank result, similar to Sal1, a single digest of pMICO (lane 3). There was an error in interpreting the protocol since the loading dye was applied after the loading process, making it invisible. It is recommended that loading dyes be added before loading samples into agarose gels to impart colour and density to the samples. In this case, the DNA will diffuse away from the gel, making enzymes unable to digest. Considering the results are blank, the Sal1 plasmid (lane 3) and the undigested plasmid (control-lane 2) of pMICO should have expected values of 6652 bp (Table 1). Lane 4 contains a single digest of pMICO, Not1, resulting in a band of approximately 6500 bp, very similar to the expected values for a single digest of the pMICO plasmid (Figure 1). Double digestion of the plasmid sample (Not1 and Sal1) yielded two bands of approximately 3500 bp and 3000 bp (Figure 1). As the expected results of the double-digested pMICO base pairs are 3718 bp and 2933 bp, this result is very similar, therefore, lane 5 contains pMICO plasmids, which provides additional evidence of similarity in observed bands and expected values. An undigested pGLO plasmid (control) on lane 6 has a band of approximately 6000 bp, like the expected value of 5317 bp. Lane 7 has a double digest of pGLO, Pst1 with observed values of approximately 4500 and 1000 bp, which are very close to the expected values of 4296 and 1074 bp (Table 1).

The DNA produced by an undigested and digested plasmid can be linear, nicked, or supercoiled when it passes through a gel. As a result of mechanical damage or cutting, additional forms may appear. Lanes 2 and 3 are blank because the loading dye was not added to the sample when it was loaded, so it is impossible to determine whether they are linear, nicked, or supercoil (see Figure 1– lanes 2 and 3). DNA in Lane 4 is linear at 6500 bp, as evidenced by the two ends on either side of the DNA molecule (see Figure 1– lane 4). Lane 5 has nicked and supercoiled DNA at 3500 bp and 3000 bp. A nicked DNA band can be seen at the top of the band in Figure 1, and two bands were formed, which both indicated that it was nicked DNA. It can also be supercoiled because its conformation causes it to migrate faster than predicted in an agarose gel. Lane 6 has linear DNA because both strands of the DNA helix were cut at the same time at 6000 bp (see Figure 1). Lane 7 is also linear DNA at 4500 bp and 1000 bp. (Tirabassi, 2017)

The laboratory’s results are generally expected, but some limitations may affect them. Since the test is conducted once, some limitations may occur. Repeating the test multiple times will yield more reliable results if the results are the same each time. In addition, the concentration for the pMICO plasmid given was just enough for performed digestion. Lastly, the gel may also contain additional DNA forms because of the rough handling of the undigested plasmid.

Conclusion:

In conclusion, isolated plasmid fragments from pMICO indicate that E. coli claiming to have the plasmid is positive since fragment sizes were consistent with what would be expected when cut with Not1 and Sal1. The expected fragment sizes were observed when the plasmid was cut from pGLO by E. coli treated with Pst1. Accordingly, the laboratory results are similar to those expected, suggesting that E. coli strain BL21 has pMICO and pGLO plasmids. Therefore, the BL21 bacterial strain is suitable for studying P. falciparum parasites.