MSP1_19: Isolation of the Vaccine Candidate Gene from

the Malaria Causing Parasite Plasmodium falciparum

Cassia Kent

FSI 2010

This project consists of two parts. Part I confirms the genetic sequence of MSP1-19, a blood stage protein. To verify that the isolated gene is the desired MSP1_19 antigen, confirmation of the genetic sequence is needed. If confirmation was not achieved, there is a likelihood the vaccine would not produce the correct antigen, making the entire vaccine futile. Part II integrates MSP1_19 into the plant expression (pSAT) vector. If the MSP1_19 gene was not integrated into the pSAT vector the production of transgenic carrots would be impossible. The reason for this is due to the fact that the pSAT vector is a very unique vector. It is one of the only plant expression vectors that can combine multiplie genes. The carrot would them not be able to express the three foreign genes without the successful ligation of the pSAT vector with the target genes. To have a three-pronged protein approach, there must be expression of the three foreign genes. So without the expression, the vaccine will lose its purpose and will not work.

The following steps are involved with both part I and II: Primer Design, PCR, Agarose Gel Electrophoresis, PCR Purification of DNA, NanoDropping, Transformation of Plasmid to JM109 Cells, Glycerol Stocks, Purify Plasmid DNA, Confirm Presence of Insert by EcoR1 Cut or PCR, Send off for Sequencing.

Primer Design Primers for three proteins, AMA1, MSP2, and MSP1_19, were designed in the laboratory (Table 1). Cassia Kent designed a primer for MSP2, Xiaoye Wang designed a primer for AMA1, and Jacqueline Johnson designed primers for AMA1, MSP2, and MSP1_19.

Primers are short, synthetic oligonucleotides that amplify your area of interest (Primer Design, 2008). They attach to the DNA sequence of a gene and provide a template strand for Taq polymerase, an enzyme that replicates DNA (Primer Design, 2008). To construct a primer, one must have a forward and reverse primer. The forward primer corresponds to the 5’ boundary of the sequence you want to amplify; it is the 5’ to 3’ sequence which corresponds to the amplified region (Keenan, 2010). The reverse primer corresponds to the 3’ boundary; it is the reverse complement of the sequence corresponding to the 3’ end of the amplification region (Keenan, 2010)

Some rules for primer design are:
1. A length of 17-25 basepairs with a melting point of 52°C-75°C (55°C-58°C produce the best results)
2. A 40-60 percent GC count
3. Having a GC clamp at the 3’ end or within the last five bases of primers (increases efficiency of priming)
4. Avoid ending primers with an A or T
5. Avoid runs (same base repeating with a max of 4) or repeats (dinucleotide combinations repeating with a max of 4) in a primer as these can result in mispriming
6. Make sure that the 3'-ends of primers are not complementary as otherwise primer dimers preferentially will be synthesized
7. Limit the number (and associated free energy) of potential dimers and secondary structures (such as hairpin loops) (Keenan, 2010)

To make sure all of these steps were abided by, an Integrated DNA Technology (IDT) calculator was used for each sequence selected. The website for this calculator is: http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/Default.aspx.

Once all criteria are met, primer sequences were sent to Davis sequencing (Davis, California) and obtained from Invitrogen (Carlsbad, California).

Amplification of purified genomic DNA. MSP1_19, AMA1, and MSP2 were all fully amplified. MSP1_19, AMA1, and MSP2 genes were PCR-amplified with primers sets (listed in Table 1).

Polymerase Chain Reaction (PCR). A PCR multiplies a specific region of genomic DNA, or primer, that is being isolated millions of times. This multiplication creates a workable quantity. To carry out a PCR, a thermocycler, which utilizes temperature cycling is used as well as a specific formulation of reactants. (Tables 2 and 3) (Figure 3). To separate (melt) the DNA, a high temperature of 94 degrees Celsius is applied (Kubista, et al., 2006). The temperature is then lowered—this lets the primers anneal to the template (Kubista, et al., 2006). After this, the temperature is set to 72 degrees Celsius—this is the optimum temperature for the polymerase, because it does the best job extending the primers by incorporating the dNTPs (Kubista, et al., 2006). Strain 3D7 genomic DNA of Plasmodium falciparum was used for all DNA isolations.

Agarose Gel Electrophoresis. To determine whether MSP1_19, AMA1, and MSP2 inserts were in the samples, agarose gel electrophoreses were ran. To prepare agarose gels, agarose powder is mixed with 1X TAE buffer (to help maintain pH), melted by heat (such as a microwave), and poured into a gel apparatus after it is partially cooled. The gels have wells in them (Figure 4), which is where the samples are placed. Electrophoreses separate macromolecules that have different size, charge, or conformation (Colorado State University, 2000). During the process, charged molecules are placed in an electric field. Nucleic acids have a negative charge due to their phosphate backbone, so therefore, they migrate towards the anode (Colorado State University, 2000). The distance the samples travel determines the number of basepairs they contain which refers to their size. To determine how many basepairs there are, the gels are compared to the ladder, which is typically placed in the first well. This ladder can then be compared to The TrackIt™ 1 KB DNA Ladder, which has predetermined DNA segments are particular sizes that run out on the gel parallel to the samples. From this, basepair length of the sample can be determined.

PCR Purification of DNA The next step was to complete PCR purifications. In a PCR, there are several proteins (TAQ polymerase and nucleotide triphosphates) and many reagents (magnesium chloride and a PCR buffer). Once PCR thermocyles have completed, the need for any of these proteins or reagents is eliminated. Following the amplification of the genes, a PCR purification is then completed to remove the unneeded compounds mentioned above. After the PCR purification, the final product is only DNA and nuclease free water. All PCR purifications were completed using a Qiagen kit specified as QIAquick PCR Purification (catalog number 28104).

NanoDropping To ensure the samples were acceptable, NanoDrop readings were conducted for MSP1_19, MSP1_19 integrated in the pGEM-T Easy vector, and pSAT cassette vectors using NanoDrop 2000 Technology. As mentioned earlier, due to a time constraint, there are no readings for AMA1 and MSP2.

For a good NanoDrop reading, the nucleic acid content must be a high number. Nucleic acid content measures how much DNA a sample contains—nanograms per microliter. The 260/280 ratio gives the purity of the DNA and dictates whether it is of good quality or not. This reading must be between 1.79 and 1.89. A 260/280 ratio reading of 1.85 is of really good quality—there is minimal, if any protein or RNA contaminating the sample.

PART I: Ligate MSP1_19 Samples into pGEM-T Easy vector using T4 DNA Ligase The purpose of this ligation was to clone gene products into pGEM-T Easy vector (Promega, Madison, Wisconsin) (catalog number 26722604). Quantities for ligations were determined (Table 4). Due to a time constraint, AMA1 and MSP2 were not able to be ligated.

PART II: Cut pSAT plasmid and inserts with EcoR1. MSP1_19 samples that were amplified at the beginning of this project and pSAT vector cassettes (1398, 1412, and 1420) were taken and cut with EcoR1. EcoR1 is an enzyme that most literally “cuts” the DNA. All of the pSAT cassettes came as a circular DNA segment and have a small segment (6 basepairs) that codes for a “cut site.” When EcoR1 recognizes a cut site, the enzyme cuts in that exact spot. Also, the MSP1_19 DNA samples were primer-designed to have EcoR1 cut sites. By both being cut, it is easier to ligate the pSAT vector with MSP1_19 DNA insert.

PART II: De-phosphorylated with Antartic Phosphate enzyme. To make sure the pSAT vector cassettes do not re-ligate to themselves, de-phosphorylation must occur. By de-phosphoylating the samples the removal of a phosphate group occurs, which disables the vector from re-ligating to itself.

PART II: Ligate pSAT vector cassettes 1398, 1412, and 1420 with MSP1_19 DNA insert. Ligation of the EcoR1 cut MSP1_19 (685 bp segment) with all of the EcoR1 cut and De-phosphorylated pSAT cassettes. Therefore, a total of three different ligation reactions occurred (MSP1_19 with 1398, MSP1_19 with 1412, and MSP1_19 with 1420).

Transformation of plasmid to JM109 cells Naked Escherichia (E.) coli JM109 cells were transformed with ligated product and were grown in Luria-Bertani (LB) medium at 37°C for DNA isolation. When required Ampicillin was used at 50 micrograms per milliliter concentration. Plasmid purification was completed using QIAgen kit specified as QIAprep spin Miniprep Kit (catalog number 27104).

PART I: Plate bacteria on AMP, IPTG, and XGAL containing LB agar plates. The pGEM-T easy vector contains a LacZ gene that if the insert is successfully integrated into the pGEM-T easy vector, a 5’ indigo product is not produced. If the ligation is unsuccessful, the LacZ remains intact and is able to utilize the compounds IPTG and XGAL to create the 5’ indigo product. This indigo product makes the colonies appear blue to the human eye. This is useful when plucking colonies because it serves as an indicator to the researcher on which colonies are positive for the MSP1_19 insert.

PART II: Plate bacteria on AMP containing LB agar plates. The pSAT and the pGEM-T Easy vectors both contain an ampicillin resistant gene in its DNA sequence. The gene allows the bacteria that take up the plasmid to produce a protein that makes the bacteria resistant to the antibiotic ampicillin. This enables bacteria to grow up on plates or in tubes that contain AMP. This is important for selection of specific bacteria that contain the only our vectors.

Glycerol Stocks. Glycerol stock solutions were made using 450 milliliters of the bacteria grown and 50 microliters of glycerol. The samples were placed in a -80 degree Celsius freezer.

Purification of plasmid DNA. Plasmid purification consolidates the DNA and ensures that there is only plasmid DNA present in nuclease free water. This is used in the confirmation steps. In the future, the pSAT plasmid will be used to transform Agrobacterium which will be used to transfect the carrot calli.

Confirm presence of insert by EcoR1 cut or PCR. This step was completed for both parts of the research. As stated above EcoR1 is an enzyme that most literally “cuts” the DNA. When doing a confirmation we utilize the natural cutting ability of the enzyme EcoR1 to cut out the insert from the pSAT vector. After which the cut samples are run on a gel electrophoresis and bands are analyzed for the present of the DNA insert.

Send off for sequencing. After all steps have been completed, the purified products will be sent to Davis Sequencing (Davis, California).