South African G9P[6] strain, for expression in several yeast strains
3.3.5 Genomic DNA isolation
172 We expected to see one band namely at 48kDa (VP6) since the group specific/VP6 antibody was used that only detects VP6. Unfortunately, no expression for VP6 in yeast could be detected as seen in Figure 3.31. The next step was to determine whether or not the ORFs of genome segment 2 and genome segment 6 were present in these recombinant yeast colonies, since it was possible that the ORFs did not integrate correctly into the different yeast strains. In order to determine whether or not the ORFs were present the same colonies (from each yeast strain used for the western blot analysis) DNA were examined.
This was achieved by plating the glycerol stocks of each recombinant yeast strain colony out on YPD agar medium and incubating overnight at 37°C (Kluyveromyces marxianus) and 30°C (Kluyveromyces lactis, Candida deformans, Saccharomyces cerevisiae, Yarrowia lipolytica, Arxula adeninivorans, Hansenula polymorpha and Debaryomyces hansenii). All further experiments were carried out from these plates.
173
Figure 3.32: Analysis by agarose gel electrophoresis of genomic DNA extraction of pKM173_VP2/6 containing colonies. (A) Lanes: 1 and 10) 10 000bp O’Generuler DNA marker. A 20 µl volume of the 50 µl extraction was loaded as follows; lanes: 2) A. adeninivorans colony 1; 3) C. derformans colony 1; 4) D. hansenii colony 1; 5) H. polymorpha colony 1; 6) K. lactis colony 1; 7) K. marxianus colony 1; 8) S. cerevisiae colony 1; 9) Y. lipolytica colony 1; 11) A. adeninivorans colony 3; 12) C. derformans colony 2; 13) D.
hansenii colony 2; 14) H. polymorpha colony 2; 15) K. lactis colony 2; 16) K.
marxianus colony 3; 17) S. cerevisiae colony 2; 18) Y. lipolytica colony 2. (B) Lane: 1) 10 000bp O’Generuler DNA marker. A 20 µl volume of the 50 µl extraction was loaded as follows; lanes: 2) A. adeninivorans colony 2; 3) K.
marxianus colony 2.
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10 11 12 13 14 15 16 17 18 3000bp
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174 DNA was obtained for all eighteen colonies as seen in Figure 3.32. All eighteen colonies of pKM173_VP2/6 recombinants, in the different yeast strains, were then screened by means of PCR, as described in section 3.2.3.5. Two PCR reactions were set up for each recombinant colony, one containing primers for the ORF of genome segment 2 and a second PCR reaction containing primers for the ORF of genome segment 6. A colony was considered positive if the PCR reaction gave an amplicon of 2700bp (for the ORF of genome segment 2) and an amplicon of 1270bp (for the ORF of genome segment 6) respectively, that could be seen on a 1% agarose gel. Figure 3.33A and B show the results of the PCR screening for genome segment 2 and genome segment 6 respectively.
175 Figure 3.33: Analysis by agarose gel electrophoresis of PCR colony screening for the ORF of genome segment 2 (VP2) and the ORF of genome segment 6 (VP6). (A) Lane: 1) 10 000bp O’Generuler DNA marker. A 10 µl volume of each 20 µl colony screening reaction of the ORF of genome segmnet 2 was loaded as follows; lanes: 2) A. adeninivorans colony 1; 3) A. adeninivorans colony 3;
4) C. deformans colony 1; 5) ) C. deformans colony 2; 6) D. hansenii colony 1; 7) D.
hansenii colony 2; 8) H. polymorpha colony 1; 9) H. polymorpha colony 2; 10) K.
lactis colony 1; 11) K. lactis colony 2; 12) K. marxianus colony 1; 13) K. marxianus colony 3; 14) S. cerevisiae colony 1; 15) S. cerevisiae colony 2; 16) Y.lipolytica colony 1; 17) Y.lipolytica colony 2; 18) positive control (pKM173_VP2/6); 19) A.
adeninivorans colony 2; 20) K. marxianus colony 2. (B) Lane: 1) 10 000bp O’Generuler DNA marker. A 10 µl volume of each 20 µl colony screening reaction of the ORF of genome segmnet 6 was loaded as follows; lanes: 2) A. adeninivorans colony 1; 3) A. adeninivorans colony 3; 4) C. deformans colony 1; 5) C. deformans colony 2; 6) D. hansenii colony 1; 7) D. hansenii colony 2; 8) H. polymorpha colony 1; 9) H. polymorpha colony 2; 10) K. lactis colony 1; 11) K. lactis colony 2; 12) K.
marxianus colony 1; 13) K. marxianus colony 3; 14) S. cerevisiae colony 1; 15) S.
cerevisiae colony 2; 16) Y. lipolytica colony 1; 17) Y. lipolytica colony 2; 18) positive control (pKM173_VP2/6); 19) A. adeninivorans colony 2; 20) K. marxianus colony 2.
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176 The results obtained from the PCR colony screening analysis for the ORF of genome segment 2 and genome segment 6 are shown in Figures 3.33A and B, respectively. It was expected that one fragment (2700bp) should result from the PCR colony screening for the ORF of genome segment 2 and one fragment (1270bp) for the ORF of genome segment 6.
The expected results were not obtained for all eighteen colonies.
Eight of the eighteen colonies screened for the presence of the ORF of genome segment 2 gave the expected result namely a band at 2700bp. These colonies were A. adeninivornas colony 1 and colony 2, D. hansenii colony 1, H. polymorpha colony 1 and colony 2, S.
cerevisiae colony 1, Y. lipolytica colony 1 and K. marxianus colony 2, as seen in Figure 3.33 A. This indicated that the ORF of genome segment 2 encoding for VP2 was only present in these recombinant yeast constructs. Seven of the eighteen colonies screened for the presence of the ORF of genome segment 6 gave the expected result namely a band at 1270bp. These colonies were H. polymorpha colony 1 and colony 2, K. lactis colony 1 and colony 2, S. cerevisiae colony 1 and Y. lipolytica colony 1 and colony 2, as seen in Figure 3.33B. This indicated that the ORF of genome segment 6 encoding for VP6 was only present in these recombinant yeast constructs. From the eighteen recombinant yeast colonies screened for the presence of the ORF of genome segment 2 encoding for VP2 and the ORF of genome segment 6 encoding for VP6, only four colonies screened positive for the presence of both genes. These colonies were H. polymorpha colony 1 and colony 2, S cerevisiae colony 1 and Y. lipolytica colony 1.
It is clear from these experiments that the ORF of genome segment 2 and genome segment 6 was present in some of the recombinant yeast constructs, therefore, expression of these proteins should probably have been possible. The next step will be to eliminate the possibilities that could have been responsible for the reason why the rotavirus proteins (VP2 and VP6) did not express in the different yeast strains. An elimination experiment that can be conducted is to see whether or not enough RNA is produced for the proteins to express.
An experiment can also be conducted to determine why some of the yeast constructs only contained one of the ORF of the genome segments and not both of the ORF of genome segment 2 and 6.