South African G9P[6] strain, for expression in several yeast strains
3.2 Materials and methods
As mentioned previously in Chapter 2 (section 2.2) the names, suppliers, and catalogue numbers of the reagents used in this study, are included in Appendix A.
3.2.1 Virus, bacterial and yeast strains used in this project
The ORFs of the wild type genome segment 2 and 6 consensus sequence derived from the viral genome (Potgieter et al., 2009) of the human rotavirus strain GR10924 (G9P[6]) was used for this part of the study. The open reading frame of genome segment 2 and genome segment 6 was purchased from Genscript and provided in the pUC57 vector by the company.
JM109 cells are a strain of Escherichia coli (E. coli), which was used for molecular cloning (discussed in Chapter 2, section 2.2.1). Eight yeast strains, Kluyveromyces marxianus, Kluyveromyces lactis, Yarrowia lipolytica, Debaryomyces hansenii, Candida deformans, Hansenula polymorpha, Arxula adeninivorans and Saccharomyces cerevisiae were obtained from the UNESCO-MIRCEN yeast culture collection at the University of the Free State for expression experiments. Saccharomyces cerevisiae was included as reference since VP2 and VP6 were successfully expressed in this yeast previously (Rodrigues-Limas et al., 2011).
3.2.2 Cloning vector
The wide-range yeast expression system was developed at the University of the Free State and is not limited to a specific yeast strain but allows for possible heterologous expression in any yeast that can be transformed (Albertyn et al., 2011). A vector pair pKM173 and pKM177 (Figure 3.1) was designed previously to allow upon fusion a dual insert vector. The wide range yeast expression system contains the 18S rRNA target sequences of Kluyveromyces marxianus to allow genomic integration into yeast cells. The selective marker for yeast expression is the hph gene conferring hygromycin B resistance. The vectors also contain the Yarrowia lipolytica TEF promoter and the Kluyveromyces marxianus
98 inulinase terminator. To facilitate sub-cloning in Escherichia coli the vectors contain the kanamycin resistance gene. The vector pair, pKM173 and pKM177, also contains the I-SceI restriction enzyme site (pKM173 one I-SceI restriction site and pKM177 two I-SceI restriction sites). The I-SceI site allows for the cassette containing the genome segment of interest (genome segment 6 cloned into pKM177 vector) to be cloned into the pKM173 vector linearized with I-SceI resulting in the dual expression vector.
Figure 3.1: The plasmid map of pKM173 and pKM177. The plasmid maps illustrate the various properties of pKM173 and pKM177. The expression system contains the 18S rRNA target sequences of Kluyveromyces marxianus to allow genomic integration into yeast cells. The selective marker is the hph gene conferring hygromycin B resistance. The vectors also contain the Yarrowia lipolytica TEF promoter and the Kluyveromyces marxianus inulinase terminator.
99 3.2.3 DNA recombinant techniques
Recombinant DNA techniques used in this study, namely polymerase chain reaction (PCR), restriction endonuclease digestion, ligation and transformation were performed according to Sambrook and Russell (2001). Commercial kits were used for plasmid isolation, gel extraction and PCR-clean-up.
The basic principles and description of the following DNA recombinant techniques have been discussed in Chapter 2 (section 2.2.3) and will therefore not be discussed again:
restriction endonuclease digestion, purification of PCR amplicons, agarose gel electrophoresis, analysis of DNA concentration and purity, gel purification of desired DNA fragments or product, ligation reactions, preparation of chemical competent Escherichia coli cells, transformation of chemical competent Escherichia coli cells and screening of colonies of transformed bacteria. However, some DNA techniques will be discussed below, namely plasmid isolation (will be discussed since a different plasmid isolation kit was used), PCR amplification of the coding sequences, dephosphorylation, long term storage of desired colonies, DNA sequencing determination, expression of proteins and analysis of protein expression.
3.2.3.1 Plasmid isolation
Propagation of bacteria containing the plasmid of interest was inocculated into 50 ml LB medium (10 mg/ml kanamycin) and grown for 16 hours at 37°C, while shaking at 180rpm.
The next day these cultures, containing the appropriate plasmid, were centrifuged at 5 000 x g for 10 minutes and the supernatants were discarded.
A commercial plasmid purification kit, QIAGEN® Plasmid Mini, Midi, Maxi, Mega and Giga, system, was used to do the plasmid purifications. The pellets of the cultures were used to isolate plasmids using the QIAGEN® Plasmid Mini, Midi, Maxi, Mega and Giga kit. The isolation was done according to the instructions of the manufacturer. This kit is designed to allow purification of ultrapure supercoiled plasmid DNA with high yields and uses an anion- exchange–based QIAGEN-tip to isolate plasmid DNA. The kit also incorporates LysesBlue which is a colour indicator that provides visual identification of optimum buffer mixing. This prevents common handling errors that lead to inefficient cell lysis and incomplete precipitation of SDS, genomic DNA, and cell debris.
The procedure consists of seven steps namely i) preparation and lysis of cell culture, ii) DNA purification by centrifugation, iii) filtration with an anion-exchange QIAGEN tip iv) medium
100 salt wash, v) precipitation of DNA, vi) wash and vii) elution. The alkaline conditions denature the chromosomal DNA and proteins. The lysis time ensures the optimum release of plasmid DNA without the release of chromosomal DNA and without exposing the plasmid DNA to denaturing conditions for too long. The lysate is then neutralized which causes denatured components to precipitate while only small plasmid DNA renaturates and stay in the solution.
The plasmid DNA binds to the QIAGEN Anion-Exchange Resin under appropriate low-salt and pH conditions and RNA, proteins, dyes, and low-molecular-weight impurities are removed by a medium-salt wash. The plasmid DNA is eluted in a high-salt buffer and then concentrated and desalted by isopropanol precipitation. The second wash step removes precipitated salt and replaces isopropanol with the more volatile ethanol, making the DNA easier to re-dissolve. Plasmid DNA was re-dissolved in 100 µl nuclease free water.
3.2.3.2 PCR amplification of the coding sequences
The amplification was performed with the use of a BioRadTM thermocycler. The coding regions were amplified in reaction mixtures containing 0.5 µM of both the forward and reverse primer (2.5 µl) (indicated in Table 3.1) and 0.5 µl template (57 ng), 10 x Takara Ex- Taq buffer (1X), Takara Ex-Taq (1.25 units/50µl), 2.5 mM dNTP and nuclease free water added to a final volume of 50µl. The negative controls contained no template.
Table 3.1: Oligonucleotide primers used in this study for PCR amplification Primer name Oligonucleotide sequence (5’
3’)
Tm (°C)
Length (bp) VP6yeast_F CAA CCT CGA GAT GGA TGT CCT
GTA C
58.6°C 25 VP6yeast_R GTC CAG CGC TTT ATT TGA CAA
GCA TGC T
61.7°C 28 VP2yeast_F CTC ACT CGA GAT GGC GTA
CAG GAA AC
60.7°C 26 VP2yeast_R GCG TCC TAG GCT ACA ATT CGT
TCA TGA T
60.3ºC 28