The isolation and identification of plasmids pMICO and pGLO from bacterial host Escherichia coli (BL-21 strain) using 0.8 % agarose gel electrophoresis.Sample Page

Aims and Backgrounds:

Humans are particularly interested in recombinant DNA technology because it has the potential to produce vaccines against serious diseases. It enables microbial cells to produce foreign proteins by re-engineering them. (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). Plasmids are small circular DNA molecules found in bacteria and other microscopic organisms. Plasmids, unlike chromosomal DNA, replicate on their own. They contain a small number of genes associated with antibiotic resistance and can be passed between cells.

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. In this case, pMICO is a strain of E. coli that contains a plasmid encoding a protein, while pGLO contains green fluorescent protein (GFP). It is estimated that P. falciparum causes more than 90% of malaria deaths worldwide, and this aspect remains a public health concern. (Zekar, L. and Sharman, T., 2022). 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. In addition, the malaria parasite has different codon preferences during translation in E. coli, which results in low levels of protein expression. This was largely resolved by introducing a tRNA-encoding plasmid (RIG plasmid) construct that recognised these rare codons in E. coli. Despite its benefits, this system has limitations when studying proteins that are toxic to E. coli. As a result, pMICO was created to express proteins cloned from P. falciparum that would otherwise be toxic to host E. coli cells. (Cinquin, O., Christopherson, R.I. and Menz, R.I., 2001).

The aim was achieved by isolating the plasmid from the bacterial host and digesting single and double before running the samples on an agarose gel. The BL-21 E. coli strain must be confirmed to contain the plasmid pMICO before further experiments can be conducted. Luria-Bertani broth (LB) was used for cultivating this bacterial culture in 50 ml (see appendix 1 for the reagent quantity needed to prepare 50 ml of LB medium). In addition, 50 μl chloramphenicol stock solution was added (see appendix 2 for calculations on adding chloramphenicol to 50 ml of LB medium). Both pMICO and pGLO can be digested by restriction enzymes at a single recognition site, usually 4-6 base pairs; thus, double digestion is necessary. The plasmid pMICO, however, has three restriction enzymes: two single digests of Not1 and Sal1 and a double digest of Not1 and Sal1. In contrast, pGLO has only one double digest, Pst1. Likewise, both plasmids required the same buffer D and were incubated at 37 °C. Also, Not1 and Sal1 can be used for double digestion, making them the most effective restriction enzymes. After the digested samples are prepared, an undigested pMICO and pGLO sample will also be analysed on a 0.8% agarose gel to determine if the observed bands are like the expected values.

Results and Discussion:

Lane 1 displays a 1kB DNA ladder from Promega. A blank result is shown for lane 2, the undigested plasmid (control) of pMICO. Similarly, Sal1 displayed a blank result for a single digest of pMICO. An incorrect interpretation of the protocol resulted in this error, as the loading dye was applied at the end of the loading process, so it was not visible. Distinct bands were observed in other undigested and digested samples containing the loading dye. This suggests that adding the loading dye before loading samples into agarose gels is beneficial. Consequently, colour and density are only imparted to a sample when density is added. Since the gel prevents DNA diffusion away from it, enzymes cannot digest DNA once it has diffused. Considering the results are blank, Sal1 and undigested plasmid (control) of pMICO have expected values of 6652 bp in length as shown in Table 1. Lane 4 contains a single digest of pMICO, Not1, which results in a band approximately 6500 bp long. The digest of pMICO should have an expected value of 6652 bp, which corresponds to the band observed on the gel (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 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 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. When isolating plasmid DNA, supercoiled DNA is the desired species. Lane 6 has linear DNA because both strands of the DNA helix were cut at the same time at 6000 bp (see Figure 1). As seen in Figure 1, lane 7 is also linear DNA at 4500 bp and 1000 bp. (R. Tirabassi, 2021).

The laboratory’s results are generally expected, but some limitations may affect them. Since the test is conducted only 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.

In conclusion, laboratory results are very similar to those expected, implying that E. coli strain BL21 carries pMICO and pGLO plasmids with a high degree of certainty. As a result, the BL21 bacterial strain is suitable for studying P. falciparum parasites. This can be seen in the plasmid fragments isolated from pMICO, indicating that the E. coli that claim to have the plasmid do have it, as the expected fragment sizes were seen when cut with the restriction enzymes Not1 and Sal1. The plasmid from the pGLO indicated that E. coli claiming to have pGLO have the plasmid, as expected fragment sizes were observed when cutting the restriction enzyme Pst1.

Table 1: A sample of the plasmids pMICO and pGLO with an undigested and digested plasmid. The pMICO plasmid contains three restriction enzymes: two single-digested Not1 and Sal1, one double-digested Not1 and Sal1, and an undigested plasmid of pMICO (control). Whereas pGLO, only has a 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.