3.3 Results and Discussion
3.3.1 Generating three SA11 recombinant bacmids
Recombinant bacmids were generated by transforming 100ng of each SA11 pFBd donor plasmids (pFBd_VP2/VP6, pFBd_VP4, and pFBd_VP7) into the E.
coli host strain, AcBACΔCC as described in Section 2.2.2. Due to the insertion into the LacZ gene within the bacmid, white colonies represent successful recombination (Section 3.2.1). However, this alone is not a full confirmation because it is possible that in some cases where the majority colonies are white, i.e., recombinant, a small sub-population may be non-recombinants (Grose et al., 2021). These non-homogenous colonies start out white but gradually develop
67 into bulls’ eyes (white colonies with a blue centre). Bulls’ eyes were not used for bacmid isolation as they would yield mixed populations of unaltered and recombinant bacmids. Fully blue colonies containing unaltered bacmids were used as negative controls and were designated as wild-type baculoviruses.
Colonies were selected for bacmid extraction as described in Section 3.2.5. The DNA concentration for the high molecular weight bacmid DNA was obtained using a NanoDrop One spectrophotometer described in Section 3.2.5. This concentration was not an absolute representation of bacmid yield, as the crude extraction method still leaves RNA fragments and salt contaminants. It was only used to ensure that bacmid extraction was successful before PCR validation was done.
pFASTBACdual_SA11_VP2/VP6
PCR analysis was performed on 30 colonies containing pFBd_SA11_VP2/VP6 to verify that recombinant bacmids were obtained and to identify colonies with a mixture of recombinant and wild-type bacmids (bulls’ eyes). The expected results for the PCR fragments are summarised in Table 11. Figure 26 shows the PCR results for the 30 VP2/VP6 colonies along with 2 wild-type colonies. Even though the 30 colonies appeared to be white during the second blue/white screening test, PCR results indicated that 14 colonies (number 2,3,4,5,6,13,21,23,25,26,27,28,28 and 30) contained a mixture of recombinant and wild type bacmids. The 14 mixed colonies yielded the expected 6705 bp amplicon and the 300 bp negative control amplicon (Section 3.2.6 Table 11).
These could not be used further in the experiment as the wild-type baculovirus would out-compete the recombinant baculoviruses.
Seven colonies (number 7,9,10,11,12,15, and 16) appeared not to contain any wild-type bacmids and were the only ones used for further testing. Figure 26, Figure 27, and Figure 28 contained a non-specific band at around 1800 bp.
Colony 31 was a blue colony selected, and colony 32 is from untransfected competent cells used as a negative control, thus the expected size of 300 bp was obtained for their amplicons.
68 One of the drawbacks of the baculovirus system is the screening of recombinant bacmids for differentiation from wild-types. The Bac-to-Bac system has several checkpoints such as antibiotic selection, blue-white screening, PCR analysis of amplicons, and plaque purification. However, several blue colonies were still obtained under gentamycin selection, such as colony 31. The blue colonies indicate that the attTn7 site in the bacmid was not interrupted even though the expression cassette has been transposed for the selection marker to work. In 2021, another attTn7 site was discovered in the bacterial chromosome at the end of the vital glmS gene (Grose et al., 2021). This suggests that the expression cassette can also be transposed into the chromosomal DNA rather than into the bacmid, as shown with colony 31.
Figure 26: Agarose gel electrophoresis of recombinant SA 11 VP2/VP6 bacmid PCR amplicon to confirm transposition of expression cassette from donor pFBd plasmid into respective bacmid DNA. The first and last lane of each row contain an O’GeneRuler DNA Ladder Mix molecular marker. For confirmation, 30 white colonies and 2 blue colonies, as a negative control, were selected for PCR. Expected PCR amplicon size of recombinant SA11 VP2/VP6 bacmid is 6,705 bp and negative control is 300 bp. Colonies selected for further use are indicated with a star (*).
69 pFASTBACdual_SA11_VP4
Twelve white colonies were selected to amplify recombinant VP4 bacmid, as can be seen in Figure 27. Colonies 1, 2, 3, 4, 5, 7, 9, 11, and 12 had no amplification of the recombinant bacmid. Only colony 6, 8, and 10 contained the correct size amplicon of the expected 5,193 bp.
Figure 27: Agarose gel electrophoresis of recombinant SA11 VP4 bacmid PCR amplicon to confirm transposition of expression cassette from donor pFBd plasmid into respective bacmid DNA. The first and last lanes contain an O’GeneRuler DNA Ladder Mix molecular marker. The expected PCR amplicon for the recombinant SA11 VP4 is 5193 bp. Poor amplification can be observed as only 3 colonies contain the correct amplicon. Colonies 6, 8, and 10 contained the correct size amplicon mark with a star (*). Colony 3 was the only sample that contained wild-type bacmid, even though no other amplification can be seen. Primer dimerization is seen as well.
pFASTBACdual_SA11_VP7
Six colonies (Colony numbers 1,2,3,4,5, and 12) contained no amplification of the recombinant cassette. However, colony 1, 5 and 12 contains the 1800 bp non-specific amplification as described above. Only colony 6, 7, 8, 9, 10, and 11 have the correct amplification of 3,843 bp for it to be recombinant VP7. No blue colony containing wild-type bacmid was used as the VP2/VP6 PCR results already contained wild-type bacmids used in all the experiments.
70 Figure 28: Agarose gel electrophoresis of recombinant SA11 VP7 bacmid PCR amplicon to confirm transposition of expression cassette from donor pFBd plasmid into respective bacmid DNA. The first and last lanes contain an O’GeneRuler DNA Ladder Mix molecular marker. The expected PCR product size for the recombinant VP7 bacmid is 3842 bp. Colonies 6 - 11 contained the correct amplicon size as expected.