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Summer Research Fellowship Programme of India's Science Academies

An Evaluation of the localization of calmodulin in p099 plasmodium falciparum (3D7)

Prangya Paramita Sahoo

Central University of TamilNadu (CUTN), Thiruvarur, TamilNadu 610005

Dr. Pawn Malhotra

International Center for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067

Abstract

Malaria, with millions of cases accounting for thousands of deaths, is a major public health problem in tropics. It is caused by Plasmodium falciparum, P. vivax, P. malariae, P. ovale and P. knowlesi of phylum Apicomplexa. Plasmodium has a complex life cycle involving both mosquito and human hosts. In humans, it invades hepatocytes and subsequently erythrocytes and undergoes several developmental transitions by asexual multiplication resulting in malaria symptoms. Parasites have developed resistance to already discovered anti-malarial drugs and hence, there is a need for discovering new drugs which can efficiently target malaria parasites. Erythrocyte invasion of the parasite is a crucial step for the parasite and a chief target for intervention. There are reports suggesting that parasites require Calcium/Calmodulin for intracellular growth and invasion. In an event of rising of intracellular calcium level, four calcium ions bind to Calmodulin (CaM) and form a Ca2+-CaM complex which binds to the target proteins for initiating various signalling cascades. CaM is a dumb-bell shaped ubiquitous eukaryotic protein which is involved in Ca2+ dependent regulations of major cellular events and the free parasites do contain CaM. Also, the growth of parasite gets inhibited by Ca2+ ionophores, Ca2+ channel blockers and CaM antagonists. Our objective was to study about the localization of CaM protein in Plasmodium falciparum strain 3D7 by expressing it in Green Fluorescence Protein (GFP) vector. CaM gene when expressed along with GFP in its upstream will emit fluorescence. With fluorescent microscope the localization of the CaM inside the cell can be visualized. The knowledge about localization can help to find cellular pathways involved in erythrocytic invasion which can be targeted for both treatment and prevention of malaria. For this we amplified PfCaM gene along with GFP primers, made copies of it, cloned the gene in GFP vector pSSPF2, amplified the number and then prepared the pDNA for transfection into the Plasmodium falciparum strain 3D7- a laboratory strain of P. falciparum.

Keywords: malaria parasite, calcium/calmodulin, erythrocytic invasion, green fluorescence protein, GFP vector, pSSPF2

Abbreviations

Abbreviations
PfCaMPlasmodium falciparum Calmodulin
  GFPGreen Fluroscence Protein 
pDNA Plasmid Deoxyribonucleic Acid 
 WHOWorld Health Organization 
 RBC Red Blood Cell 
 PLCPhospholipase-C 
PfPKBPlasmodium falciparum Protein kinase-B 
 cAMP Cyclic Adenosine Monophosphate  
cGMP Cyclic Guanosine Monophosphate 
  NMRNuclear Magnetic Resonance 
  cytCytoplasm 
 mRNA Messenger Ribonucleic Acid 
PCR Polymerase Chain Reaction 
  EtBrEthidium Bromide 
  MgCl2Magnesium chloride 
  dNTPDeoxy Nucleiotide triphosphate 
  RTRoom Temperature 
  LBLuria-Bertani 
RPMRevolutions per Minute 
 MQ Milli-Q 
  EDTAEthylenediaminetetraacetic Acid 

INTRODUCTION

Background

Since the time of evolution, malaria is still continuing to remain as the deadliest disease, killing approximately half a million of people each year. With an estimated value of 219 million cases and 435000 deaths in the year of 2017 malaria was again proved to be the deadliest of all the oldest known diseases ​("Malaria, 2019​. It is caused by the Plasmodium parasite of phylum Apicomplexa and is transmitted from one person to the other by the bite of infected female Anopheles mosquito. The causative agents of malaria are Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi. Of all these aetiological agents P. falciparum and P. vivax are the ones responsible for severe malaria and P. falciparum is mostly accountable for cerebral malaria. Approximately 3.2 billion people are at risk of malaria globally. Of these, 1.2 billion people are at a higher risk of infection​(2019)​ ​

Malaria parasite has an intricate life-cycle, involving an invertebrate mosquito vector and a eukaryotic parasite. The complexity of the situation of malaria has increased in recent years because of the increasing resistance of the Plasmodium parasites to the general anti-malarial drugs, and also the vector Anopheles have developed resistance to the existing insecticides. Hence, there is a dire need for new and effective anti-malarial drugs as well as vaccines for preventing the same.

During infection parasites invade the hepatocytes and the erythrocytes in the human host. Calcium, as well as parasite calmodulin play a crucial role in host cell invasion and intracellular growth​Vaid, et al, 2007​​. From a study on erythrocyte binding assay, it has been found that calmodulin is having a role in the erythrocytic invasion. GFP when present upstream of CaM, will emit fluorescence hence, the localization of CaM in the cell and its interactive pathways can be deduced and can be targeted.

Malaria parasite has an intricate life-cycle, involving an invertebrate mosquito vector and a eukaryotic parasite. The complexity of the situation of malaria has increased in recent years because of the increasing resistance of the Plasmodium parasites to the general anti-malarial drugs, and also the vector Anopheles have developed resistance to the existing insecticides. Hence, there is a dire need for new and effective anti-malarial drugs as well as vaccines for preventing the same.

Objectives of the Research

Cloning of CaM gene into GFP vector pSSPF2 and Preparation of DNA for transfection into Plasmodium falciparum 3D7.

Capture2.JPG
  • 1
Vector
map of pSSPF2:pSSPF2 has two units for gene expression in the malaria parasite. The first is composed of the P. falciparum heat shock protein 86 promoter region (hsp86-5´-red) and the 3´ sequence of the P. berghei DHFR-TS gene (PbDT-3´-pink), is for expressing the gene recombined between the BglII and PstI (or XhoI) sites. The P. falciparum calmodulin promoter (CAM-5´-blue), human DHFR gene (hdhfr-magenta) and the 3´ sequence of the P. falciparum histidine-rich protein 2 gene (hrp2-3´-cyan) compose the second expression unit for selecting successful transfectants under drug pressure with the anti-folate WR99210. These two expression units are arranged in head-to-head orientationon either side of the 0.8 kb DNA sequence containing the Rep20 repeats (Rep20-yellow). An arrow in the circle indicates the direction of transcription in each expression unit. The plasmid backbone was derived from the E. coli vector pGEM.
Sato, et al, 2003
Vector map for CaMpSSPF2 .jpg
    Vector
    map for CaM/pSSPF2
    (Partial): The gene expression of CaM-GFP is carried out by two units in the malarial parasite. The first unit is for expressing the recombined gene of interest, PfCaM, between BglII and AvrII sites. The GFP tag (GFP; green) is downstream of the gene of interest between AvrII and XhoI (or PstI) sites. Das, et al, 2012

    Scope

    Preventing and treating malaria is one of the greatest challenges for biologists. The knowledge of localization of CaM can help us to know about its resident organelle and the interacting pathways. CaM of the parasite shares 89% homology with that of mammalian CaM​​Robson, et al, 1993​​. Therefore, it cannot act as a drug target but can be studied to decipher its role in parasite invasion of erythrocytes and hence discover novel antigens participating in various pathways.

    LITERATURE REVIEW

    Information

    Since beginning of the mankind malaria has killed more people than that of any war and still now it is continuing to be one of the deadliest and challenging of all. It is transmitted by the bite of infected female Anopheles mosquito. The major malaria parasites are Plasmodium falciparum, P. vivax, P. malariae, P. ovale and P. knowlesi. Although the number of malaria cases has reduced from 239 million cases in 2010 to 219 million cases in the year 2017​​​("MalarialSite", 2019)​​, significant progress in reducing the global impact of malaria is not evident. P. falciparum was accountable for 99.7% of malaria cases in WHO African region, 62.8% in WHO South-East Asia, 69% of the Eastern Mediterranean and 71.9% of the Western Pacific; and P. vivax was accountable for 74.1% of cases in WHO Region of the Americas​​("Fact sheet about Malaria, 2019)​​​​. The increasing resistance of the parasite to most of the anti-malarial drugs is now a major challenge for scientists across the globe.

    The life cycle of the malaria parasite

    1. An infected female Anopheles mosquito carrying the sporozoites stage of the malaria parasite injects the parasites into the bloodstream while taking a blood meal. The sporozoites then travel in the blood around the body until they invade the liver hepatocytes.

    2. In the hepatocytes, the sporozoites undergo a phase of asexual multiplication known as exoerythrocytic schizogony to produce millions of uninucleate merozoites for over 5-16* days. Some parasite species of malaria also produces hypnozoites in the liver that remain dormant for an extended period, causing relapses in weeks or months later.

    3. The merozoites re-enter the bloodstream and initiate a second round of asexual multiplication phase in RBC, known as erythrocytic schizogony where they develop into mature schizont, ruptures RBC and releases merozoites which then reinvade fresh RBCs. This cycle of invasion and cell rupture repeats every 1-3 days* and results in thousands of parasite-infected RBCs in the host bloodstream, leading to illness and complications of malaria that can last for months if not treated.

    4. During the disease progression, some of the merozoites leave the cycle of asexual replication and develop into sexual forms of the parasite, called male and female gametocytes that circulate in the peripheral blood stream. In some malaria species, young gametocytes sequester in the bloodstream.

    5. When a mosquito bites an infected human, it ingests the gametocytes. Within the mosquito, these gametocytes further develop into mature sexual forms called gametes. The male and female gametes fuse to form motile diploid zygotes called ookinete in the lumen of mosquito gut, which burrow through the mosquito midgut wall and form oocyst on the other side. Thus, the process of sporogony begins.

    6. Growth and division of each oocyst produce thousands of active haploid forms called sporozoites. After 8-15 days*, the oocyst bursts, releasing sporozoites into the body cavity of the mosquito, from which they travel to and invade the mosquito salivary glands. The cycle of human infection re-starts when the mosquito takes a blood meal, injecting the sporozoites from its salivary glands into the human bloodstream.

    *time-frame depends on the malaria parasite species.

    malaria life-cycle.jpg
      Life cycle of malaria parasite​​("Malaria, 2019

      Role of Calcium in Parasite Invasion

      Although the importance of calcium ​McCallum-Deighton N and Holder AA, 1992​ as well as calmodulin has been implicated in erythrocyte invasion by Plasmodium, the identity of signalling pathways and underlying mechanisms have remained largely unknown. Various reports demonstrated that PLC-mediated control of calcium release is important for merozoite invasion. Previous studies indicate that CaM may be involved in invasion, and localization of CaM at the apical end of merozoites may facilitate this process ​Matsumoto Y, et al, 1987​. PfPKB is one of the very few CaM targets to be identified in P. falciparum ​Vaid A and Sharma P, 2006​, which corroborates well with its proposed role in the invasion which indicates that the PLC-calcium-calmodulin pathway can control invasion via PfPKB. Calcium is also considered important for the regulation of motility and invasion by Apicomplexan parasites ​​L.H. Miller, et al, 1994​​.. Therefore, phosphorylation of motor complex proteins by calcium-dependent signalling pathways may be an important step in the invasion. However, the exact mechanism of parasite invasion facilitated by calmodulin and calmodulin-like proteins are still not clear.​

      Calmodulin

      Calcium is an important second messenger, which participates in signalling cascades that regulate protein secretion, gene expression and development in eukaryotic cells Dawn A and Singh S and More KR and Siddiqui FA and Pachikara N and Ramdani G and Langsley G and Chitnis CE, 2014. High and sustained concentrations of calcium in the cytosol are lethal; thus, calcium homeostasis is tightly regulated by calcium pumps, calcium buffering proteins and intracellular organelles with different carriers. Calmodulin is an intracellular calcium receptor found ubiquitously in eukaryotes. It is capable of regulating the biological activities of many cellular proteins and transmembrane ion transporters in a calcium-dependent manner. When the intracellular calcium level rises, four calcium ions bind to CaM and Ca2+-CaM complex binds the target proteins, initiating various signalling cascades.

      CaM is encoded by a single gene and the putative protein is highly homologous to calmodulin from the other eukaryotes. The CaM gene is encoded by chromosome 14 and contains a single intron of 506bp that has the appropriate donor and acceptor splice sites Robson KJ, 1993.

      The binding of Calcium ions to CaM in the presence of cyclic nucleotide phosphodiesterase and adenyl cyclase leads to its subsequent activation and modulation of cAMP and cGMP levels. The importance of calcium in signal transduction is well established, and its indirect effects on the cell are widespread including cell cycle arrest, regulation of meiosis and regulation of transcription factors ​Harrison DG and Long C, 1968​. The calcium binding protein, CaM, is an important mediator of calcium signalling and is required for the regulation of a wide variety of Ca2+-dependent enzymes. Calcium is a second messenger that functions in signal transduction pathways in eukaryotic organisms. Ca2+ signalling has been implicated in governing myriad biological processes as broad as fertilization and development, exocytosis and muscle contraction, and transcription and chromatin remodelling in multicellular eukaryotes ​McCallum-Deighton N and Holder AA, 1992​.

      CaM is a dumbbell-shaped protein, with two globular Ca2+ binding domains connected by long exposed alpha-helix. It has two EF-motifs (helix-loop-helix) at one end and four EF-motifs in all. Each EF-motif has a loop of 12 amino acids, rich in aspartic acid and glutamic acids side chains which form an ionic bond with Ca2+. Therefore, each globular end binds with two calcium ions. It has been found that carbonyl-terminal has ten times more affinity for Ca2+. These EF-motifs change conformation upon binding calcium ions. Each EF-hand motif contains two alpha-helices connected by a 12-residue loop. A calcium ion binds to the loop region and changes the relative positions of the alpha helices. In the absence of calcium, the alpha-helices in the EF-motifs of calmodulin are positioned almost parallel to each other. This is known as the closed conformation. Upon binding calcium, calmodulin undergoes large conformational changes. The crystal structure of calcium-loaded calmodulin exhibits a dumbbell shape. However, it was later found that part of the central linker region of calcium-bound calmodulin is flexible in solution. Both X-ray and NMR studies agree that alpha helices of EF-hand motifs change their position relative to each other, forming almost perpendicular conformations (open conformations). This radical change allows calmodulin to increase its binding affinity for a number of target proteins.

      It has been shown through numerous structural studies that upon binding its target peptides, calmodulin form compacts globular conformations by bending its central helix. The high content of methionine residues (9 out of 148 residues) in calmodulin has been believed to be responsible for the ability to bind numerous target proteins. Indeed 8 of the 9 methionine are directly involved in binding to all target peptides studies so far by X-ray and NMR.

      CaM showing dumbbell-shape.png
        CaM showing dumbbell-shape

        P. falciparum and Calmodulin

        Previous work carried out with human malaria parasites (P. falciparum) showed that it is able to maintain levels of [Ca2+]cyt between 40nM and 100nM during their intraerythrocytic maturation Read LK and Mikkelsen RB, 1990. The invasion of the parasite, as well as its development inside the host cell, depends on the availability of free Ca2+ in the extracellular medium Wasserman M and Alarcón C and Mendoza PM, 1982. The parasite growth is inhibited by a variety of Ca2+ ionophores, Ca2+ channel blockers and calmodulin antagonists Tanabe K and Izumo A and Kato M and Miki A and Doi STanabe K and Izumo A and Kato M and Miki A and Doi SStage-dependent inhibition of Plasmodium falciparum by potent Ca2+ and calmodulin modulators.. 36,

        Plasmodium CaM possesses high identity (89%) with the gene of mammals Robson KJ, 1993, is encoded on chromosome 14 and contains a single intron of 506 base pairs. Besides CaM, the Plasmodium genome encodes for CaM-like proteins and putative CaM proteins, which are referred to as CaM-related proteins Vaid, et al, 2007. There are studies showing inhibition of erythrocyte invasion when PfCaM antagonists were used because it inhibited the growth of protozoa itself. This indicates that there must be a crucial role of protein CaM in malarial parasite survival.

        By radioimmunoassay study, it has been found that the free parasites do contain CaM and in schizont-infected erythrocytes the level of CaM was 23.3 ± 2.7 ng per 106 cells while that of normal it was 11.2 ± 1.5 ng per 106 cells, suggesting that free Ca availability is crucial for erythrocyte invasion and its levels were proportional to parasite maturity. By immunoelectron microscopy, it was found that CaM diffuses within the cytoplasm of mature parasites and at the apical end of merozoites inside the ductule of rhoptries Scheibel LW and Colombani PM and Hess AD and Aikawa M and Atkinson CT and Milhous WK, 1987 .

        Significance of the study

        Our lab has identified PfCaM as an RBC binding protein. For understanding its localization as well as its interaction with other pathways, we have prepared a plasmid containing CaM in the downstream of GFP, so that its residing organelle can be traced after transfecting it into the Plasmodium falciparum strain 3D7.

        METHODOLOGY

        PCR amplification of PfCaM gene with pSSPF2 specific primers from cloned expression vector pET28a

        PCR procedure was carried out for Calmodulin gene with pSSPF2 specific primers in a reaction volume of 50µL with Q5 polymerase enzyme (2 samples 50µL each).

        PCR composition for the amplification of PfCaM gene
        STOCK SOLUTION & CONCENTRATION WORKING CONCENTRATION VOLUME (in µL) 
         MQ Water - 34.75
         5X Q5 Reaction Buffer0.5X 5 
          25mM MgCl2  1.5mM3 
         10mM dNTPs0.2mM 1 
         Forward Primer (20µM)0.4µM1 
         Reverse Primer (20µM)0.4µM 1 
         Q5 Polymerase (5000U/mL)  0.025U/µL0.25 
        Template (pET28a- PfCaM)  - 4
        Total Volume   - 50 
        PCR condition for the amplification of PfCaM gene
         TEMPERATURE (°C)CONDITION  DURATIONNo. of Cycles 
         95Initial Denaturation 10 min1
         95 Denaturation50 sec30
         45 Annealing90 sec30
         68 Extension1 min30
         72 Final Extension5 min1
         4 Cooling1

        PfCaM Genomic Sequence (Introns highlighted) (982 bp; Introns: 532 bp)

        ATGGCAGACAAGTTAACAGAAGAACAAATTTCGGAATTCAAAGAAGCCTTTAGTTTGTTTGATAAAGATGGAGATGGAAGTAAATCATAAAAAAGAAAGAAAAAAATGAACATATTATATATATATATATATATATATATATATTTATATATTTAAACATTGTTTATAATATGTAATTGTTCCTTTTCTTTTTAACAATTTCATATTTTAATGATTTAATATTCCTATTGAATCTTTTTATTTCTTATGTATATATGAAATTTATATAAAAATTATATTATATATTACCTGAACAGTTCATAAAAATATATATATATATATATATATATACTTTTTTGGCTGTAAAACGTGAACAAAAAAAAATATTATGAACAATTCAGACAAATTAAATGAAAATGAAAGAAAAAATTAAAACGTTATAAAAATGTTATGTCCAAATAGCCAAAAATATAAATCATATTGATGTAGTACATATAAAAATATACATATATATATATATATATATATATATATATATATATATATATATATATATCAATACATACAGTTCTTATTTTATATTATATTATATTATATTATATTATATTATATTTTTTTATTTTTCTCATTAGCTATAACAACTAAGGAGTTAGGAACGGTCATGAGATCTTTAGGACAAAATCCAACTGAAGCAGAATTGCAAGATATGATTAATGAAATTGATACAGATGGGAACGGAACGATCGATTTTCCCGAATTTCTAACCTTAATGGCAAGAAAATTAAAAGATACGGACACTGAAGAAGAATTAATTGAAGCCTTCCGAGTTTTTGATAGAGATGGTGATGGATATATAAGTGCAGATGAACTAAGGCATGTCATGACAAATTTGGGAGAAAAATTAACAAATGAAGAAGTTGATGAAATGATAAGAGAAGCTGATATTGATGGTGATGGACAAATTAATTATGAAGAGTTTGTTAAAATGATGATAGCCAAATGA

        Predicted RNA/mRNA Sequence (Introns spliced out) (450 bp)

        ATGGCAGACAAGTTAACAGAAGAACAAATTTCGGAATTCAAAGAAGCCTTTAGTTTGTTTGATAAAGATGGAGATGGAACTATAACAACTAAGGAGTTAGGAACGGTCATGAGATCTTTAGGACAAAATCCAACTGAAGCAGAATTGCAAGATATGATTAATGAAATTGATACAGATGGGAACGGAACGATCGATTTTCCCGAATTTCTAACCTTAATGGCAAGAAAATTAAAAGATACGGACACTGAAGAAGAATTAATTGAAGCCTTCCGAGTTTTTGATAGAGATGGTGATGGATATATAAGTGCAGATGAACTAAGGCATGTCATGACAAATTTGGGAGAAAAATTAACAAATGAAGAAGTTGATGAAATGATAAGAGAAGCTGATATTGATGGTGATGGACAAATTAATTATGAAGAGTTTGTTAAAATGATGATAGCCAAATGA

        Predicted Protein Sequence (149 aa)

        MADKLTEEQISEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELQDMINEIDTDGNGTIDFPEFLTLMARKLKDTDTEEELIEAFRVFDRDGDGYISADELRHVMTNLGEKLTNEEVDEMIREADIDGDGQINYEEFVKMMIAK

        Molecular weight: 16931Da

        Isoelectric Point: 3.86

        Episomal Primers (GFP):

        Vector: pSSPF2

        Forward

        5’ GGATCCAGAAATATATCAATGGCAGACAAG 3’ BamHI Tm: 53C; 70.0ᵒC

        Reverse

        5’ CCTAGGTTTGGCTATCATCATTTTAACAAAC 3’ AvrII Tm: 51C; 68.0ᵒC

        PCR purification of PfCaM gene using QIAGEN’s QIAquick gel extraction kit

        2µL of PCR product along with 2µL of dye was resolved on a 1% agarose gel containing 0.5µg/mL of EtBr for checking the amplification of PfCaM gene.

        To the remaining PCR products, 300µL of QG buffer was added, mixed well and incubated at 50°C for 10 min. Samples were carefully loaded onto a QIAquick spin column and centrifuged for 1 min at 13K rpm. Flow-through was collected and again loaded onto the QIAquick spin column and centrifuged for 1 min at 13K rpm. This step was repeated twice. Flow-through was discarded and to the column 300µL of PE buffer was added. The column was allowed to stand for 2-3 min and then centrifuged for 1 min at 13K rpm. The flow-through was discarded and dry spin was performed for another 5 min at the highest speed. The column was then transferred to a fresh 1.5mL centrifuge tube and kept opened for 2 min at RT to allow PE buffer ethanol to evaporate. 50µL of preheated MQ water was added onto the centre of the column and allowed to stand for 2 min at RT and centrifuged at 13K rpm for 1 min. Flow-through was collected and loaded again onto the column and centrifuged at 13K rpm for 1 min. The column was discarded and the concentration of the flow through was checked using Nanodrop reader.

        Ligation of PCR amplified products into pJET1.2/blunt (cloning vector)

        Ligation mix was prepared and kept for 30 min incubation at RT (The molar ratio of insert to vector DNA used was 3:1).

        Ligation Mix pJET and PfCaM
        Vector pJET  1µL
         PCR Product 1µL
         2X pJET Buffer 5µL
         T4 DNA Ligase Enzyme1µL 
          Total Volume 8µL

        Transformation of competent cells DH10β

        Competent cells DH10β were taken from -80°C and thawed on ice for 3-5 min. Complete ligation mixture was added and tapped gently to mix, incubated in ice for 30 min. Heat shock was given to the cells at 42°C for 90 sec in water bath and vial was incubated in ice for 5 min. Then, 1 mL of LB broth was added to the vial and incubated in horizontal shaker incubator at 37°C for 90 min at 150 rpm. The vial was then centrifuged at 5K rpm for 5 min and 800µL of the supernatant was discarded. The pellet was dissolved in remaining supernatant and plated onto LB-agar plates containing 100µg/mL of Ampicillin and incubated at 37°C overnight. Colonies obtained were streaked to prepare the master plate.

        Isolation of recombinant plasmid

        A single positive colony was picked up from the plate and was inoculated in 10 mL of LB broth having Ampicillin at a working concentration of 100µg/mL and grown overnight at 37°C shaker. Plasmid DNA was isolated using HiYield Plus Plasmid Mini Kit (Real Biotech Corporation). Cells were harvested by centrifugation at 13K rpm for 2 min at RT. The bacterial cell pellet was resuspended in 200µL of resuspension (PD1) solution. 200µL of lysis (PD2) solution was added and mixed by inverting the tubes. 300µL of neutralization (PD3) solution was added and mixed by inverting the tubes. Mixture was centrifuged at 13K rpm for 20 min at RT. Supernatant was transferred onto a fresh eppendorf and centrifuged at 13K rpm for 10 min at RT. Supernatant was loaded onto the QPD column and centrifuged at 13K rpm for 1 min at RT. Flow-through was loaded onto the column and centrifuged at 13K rpm for 1 min at RT. This step was repeated twice and flow through was discarded. W1 solution was added to the column and centrifuged at 13K rpm for 1 min at RT. The flow-through was discarded and the column was washed with 600µL of Wash solution and centrifuged at 13K rpm for 1 min at RT. The bound plasmid DNA was eluted by adding 50µL of preheated autoclaved MQ water to the column and centrifugation at 13K rpm for 2 min at RT.

        Restriction digestion of pJET-PfCaM

        Digestion mix of 50µL was prepared and was kept for 2-3 hrs in 37°C water bath.

        Digestion Mix pJET-PfCaM
         Template pJET- PfCaM10µL 
         BamHI  1µL
         AvrII 1µL
          10X FD Green Buffer 5µL
          MQ Water 33µL
          Total Volume 50µL

        Restriction digestion of pSSPF2

        Digestion mix of 50µL was prepared and was kept for 2-3 hrs in 37°C water bath.

        Digestion Mix pSSPF2
         pSSPF2 DNA10µL 
          BglII 1µL
          AvrII 1µL
          10X FD Green Buffer 5µL
        MQ Water  33µL
          Total Volume 50µL

        Gel Extraction of PfCaM gene and pSSPF2 vector backbone using QIAGEN’s QIAquick gel extraction kit

        Digested pJET and pSSPF2 DNA were resolved in 1% agarose gel containing 0.5 µg EtBr, along with 1Kb DNA ladder and the expected sized band was excised from the gel. Fallouts were then purified using QIAGEN’s QIAquick gel extraction kit.

        Ligation of gel extracted PfCaM product into gel extracted pSSPF2/sticky (GFP vector)

        Ligation mix of 20µL was prepared and kept for overnight incubation at 16°C water bath (The molar ratio of insert to vector DNA used was 3:1).

        Ligation Mix pSSPF2-PfCaM
         Vector pSSPF2 5µL
        PfCaM 6µL
        10X Reaction Buffer  2µL
        T4 DNA Ligase Enzyme  1µL
         Double Distilled Water  6µL
          Total Volume 20µL

        Transformation of competent E. coli Top 10 cells and isolation of recombinant plasmid

        Competent E. coli Top 10 cells were taken from -80°C, thawed on ice for 3-5 min. The complete ligation mixture was added, mixed and then transformed. The recombinant plasmid was then isolated.

        Screening for positive pSSPF2-PfCaM clones by double digestion and checking the cassettes of vector pSSPF2

        Digestion mix of 20µL was prepared and was kept for 2-3 hrs in 37°C water bath.

        Digestion Mix for screening positive pSSPF2-PfCaM
         pSSPF2-PfCaM DNA2µL 
          Enzyme 10.5µL 
          Enzyme 2 0.5µL
          10X FD Green Buffer2µL 
          MQ Water 15µL
          Total Volume 20µL

        Midi isolation of positive pSSPF2 using FastIon Plasmid Midi Kit (Real Biotech Corporation)

        A single positive colony having all the cassettes of pSSPF2 as well as PfCaM, was picked up from the master plate and was inoculated in 200 mL of LB broth with 200µL of Ampicillin and grown overnight at 37°C shaker. The cells were then harvested at 6K rpm for 5 min. The PI column was equilibrated with 5 mL of PEQ buffer by gravity flow. The pellet was resuspended with 6mL of PM1 buffer and to that 6mL of PM2 buffer was added and mixed by inverting. The solution was allowed to stand for 2 min and then 6mL of PM3 buffer was added to that and mixed immediately by inverting the tube. The mixture was then centrifuged at 10K rpm for 30 min. The supernatant was then carefully transferred to a fresh 50mL falcon tube and centrifuged at 10K rpm for 10 min. The supernatant was then transferred on to the equilibrated PI column and allowed that to pass through gravity flow. The flow-through was collected and again loaded onto the column and allowed that to pass by gravity flow and the step was repeated twice. The flow-through was then discarded, 12mL of PW buffer was added onto the column and allowed to empty completely by gravity flow. The column was then placed in a fresh 50mL falcon tube, 8mL of PEL buffer was added and allowed to empty completely by gravity flow. The column was then discarded and to the flow through (pDNA) 6mL of chilled Isopropanol was added, mixed and centrifuged at 10K rpm for 90 min at 4°C. The supernatant was discarded, the pellet was washed with 5mL of 75% ethanol and centrifuged at 10K rpm for 30 min at 4°C. The supernatant was discarded and the area of the falcon where the pellet was seen was marked. The pellet was dried in 37°C incubator (overnight incubation). The pellet was then dissolved in 200µL of preheated autoclaved MQ water. The purity of the pDNA was checked on 1% agarose gel.

        Preparation of DNA for transfection (In Culture Hood)

        Eluted DNA (200µL) was precipitated with 0.1 volume of 3M Sodium Acetate and 5 volume of 100% Ethanol (Merck). The mixture was kept at -80°C overnight and then centrifuged at 13K rpm for 30 min. The supernatant was discarded, the pellet was washed with 1mL of 75% ethanol and centrifuged at 13K rpm for 15 min (Repeated twice). The plasmid was air dried in the culture hood and resuspended with 60µL of ultrapure water.

        RESULTS AND DISCUSSION

        PCR amplification of PfCaM gene

        PfCaM gene was amplified by PCR using GFP primers with pET28a expression vector as template, and resolved in 1% agarose gel. Fallout was seen as bright band in both the cases and was at expected size i.e. at 450bp. Thus, it was concluded that PfCaM gene has been amplified.

        1.JPG
          PCR amplification of PfCaM gene with pET28a expression vector as template. Amplification was done  using pSSPF2 specific primers

          Gel excision of PfCaM gene and pSSPF2 vector backbone

          PfCaM gene from pJET plasmid and pSSPF2 vector backbone were excised from agarose gel after performing double digestion with appropriate restriction enzymes. pSSPF2 vector backbone fallout appeared at 7.2kb and PfCaM fallout was seen at 450bp.

          2.JPG
            Gel excision of PfCaM gene and pSSPF2 vector backbone obtained after restriction digestion using BamHI, BglII, AvrII

            Screening for positive pSSPF2-PfCaM clones by double digestion and checking the cassettes of vector pSSPF2

            Genes present in the pSSPF2 are crucial for the survival of parasite in presence of antibiotic WR99210. Hence, by following the vector map appropriate double digestion was performed. Fallout came at the appropriate sites. Thus, midi isolation was done from the positive colony containing all the necessary genes of pSSPF2 as well as our gene of interest i.e. CaM.

            digestion.jpg
              Screening for positive pSSPF2-PfCaM clones by double digestion and checking the cassettes of vector pSSPF2
              Enzymes with expected fallout Size
              Sl. No.  ENZYMESFRAGMENT EXPECTED SIZE (bp) 
              1 XhoI, AvrII GFP 700 
              2 AvrII, BamHI CaM+hsp86-5'+Rep 20+CAM-5'2750 
              3BamHI, XhoI GFP+CaM+hsp86-5'+Rep 20+CAM-5' 3450 
              4 XhoI, HindIIIGFP+CaM+hsp86-5'+Rep 20+CAM-5'+HdHfr 3950 
              5BamHI, HindIII HdHfr/BSD 500 
              6NotI, EcoRI pGEM  3000
              7EcoRI, HindIII Hrp2-3'600 
              8XhoI, NotI PbDT-3 800 

               Confirmation of plasmid midi isolation of positive pSSPF2

              After performing plasmid midi isolation of positive pSSPF2_CaM clone, the concentrated sample was diluted ten folds and 2µL of it was loaded onto 1% agarose gel. No genomic DNA or RNA contamination was seen and the band came at appropriate size. This concluded that plasmid is pure and can be used for transfection.

              3.JPG
                Confirmation of plasmid midi isolation of positive pSSPF2

                CONCLUSION

                Hundreds of proteins are believed to be involved in malaria infection. Since, CaM is a ubiquitous protein, there must be some role of this protein in this infection, trivial or significant. There are studies showing that calmodulin is indispensible for in vitro growth and invasion of erythrocytic stages of the human malaria parasites Plasmodium falciparum but, the exact pathway of CaM interaction is still unknown hence, grabbed our interest to begin with CaM gene.

                We still have to transfect the Plasmodium falciparum strain 3D7 with the prepared pSSPF2 plasmids so that, the localization of CaM in the parasite and its interaction with other vital pathways can be known. This study might help the scientist to design drugs for targeting those pathways or the organelles where it is localizing.

                PfCaM contain an internal BglII site, hence the exact fallout of 450bp cannot be seen. For overcoming this challenge other sites along with CaM was digested for verifying the presence of the gene of interest.

                ACKNOWLEDGEMENTS

                I would like to express my sincere gratitude to INSA-SRF programme for giving me such a wonderful opportunity and to Dr. Pawan Malhotra of Malaria Biology Lab, ICGEB, New Delhi for giving me a chance to work in his lab. I’m thankful to all the scholars of Dr. Malhotra, especially Mr. Vaibhav Sharma and Ms. Sadaf Parveen for their valuable guidance and help during the tenure. I’m also thankful to my fellow trainee Thamarai Selvi for her assistance during my stay at New Delhi.At last I would like to thank my family for their constant encouragement and support.

                APPENDICES

                • Ampicillin (100mg/mL): - 1g of Ampilcillin Sodium salt was dissolved in autoclaved double distilled water and made upto a final volume of 10mL. The solution was filter sterilized and stored in 1mL aliquotes in -20°C.
                • Sodium Acetate, 3M: - 40.8g of Sodium Acetate (MW- 136.08) dissolved in 80mL of deionised water. pH adjusted to 5.2 using Glacial acetic acid. Total volume was made upto 100mL with water.
                • 50X TAE Buffer
                  • Tris Base- 242gm
                  • EDTA- 18.61gm
                  • Glacial Acetic Acid- 57.1mL
                  • MQ Water- made upto 1L

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