TITLE: Development of a reporter line for assessing the changes in gene expression of PfSir2a 
AUTHORS: Linda Anagu

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>A luciferase reporter line of the malaria parasite, </span><span style = "font-style:italic;">Plasmodium falciparum</span><span>, was designed in an attempt to measure the activity of </span><span style = "font-style:italic;">PfSir2a</span><span> promoter independent of mRNA expression in response to stress. This can also be used to isolate any promoter activity-associated-stress response from in-parasite cellular feedback attempting to restore homeostasis due to an altered sirtuin mRNA level. Here, a luciferase reporter gene was cloned under the presumptive </span><span style = "font-style:italic;">Sir2a </span><span>promoter (~1.7kb of the gene’s upstream sequence) and the reporter gene was transfected into 3D7 parasites. Its expression profile was similar to that of the endogenous </span><span style = "font-style:italic;">Sir2a</span><span> gene across the intraerythrocytic cycle. Luciferase reporters can be used to investigate the promoter activity of other genes.</span></div></div>

[STEPS]
?. [Cloning of reporter luciferase gene under PfSir2A promoter]
Extraction and Digestion of the Vector Backbone : The vector pLNSir2aGFP (or pLNSir2AGFP) with the GFP gene under PfSir2A promoter (reffered to as pLNSG vector here) and containing the AmpR gene was amplified by cloning this vector into competent E. coli (PMC103 strain, ATTC) via electroporation.
?. [Cloning of reporter luciferase gene under PfSir2A promoter]
The plasmid map of this vector (shown in the description section) was viewed in SnapGene software  (GSL Biotech LLC) so as to identify the restriction enzymes needed to linearize the vector and remove the GFP gene.
?. [Cloning of reporter luciferase gene under PfSir2A promoter]
25 µl of gently thawed  competent cells was mixed with 1 µl of this plasmid. This mixture was left    ice for 15 min and then transferred into an ice cooled
on ice
on ice
?. [Cloning of reporter luciferase gene under PfSir2A promoter]
The cuvette content was electroporated (with a GenePulser II electroporator (Bio-Rad)) at 1.8 kV and the culture recovered in 300 µl of warm  SOC broth for 1 h in a shaking incubator at
37 °C
37 °C
?. [Cloning of reporter luciferase gene under PfSir2A promoter]
The recovered culture was centrifuged at 6000 rpm for 1 min to form a pellet. 200 µl of the supernatant was discarded. The remaining supernatant was mixed with the pelleted culture.
?. [Cloning of reporter luciferase gene under PfSir2A promoter]
The mixture from 1.4 was added to 5 ml of LB (Luria-Bertani) broth (Fisher Scientific, UK) containing 5 µl of 100 mg/ml sterile ampicillin solution.  This was incubated at  overnight in a shaking incubator.
37 °C
?. [Cloning of reporter luciferase gene under PfSir2A promoter]
The pLNSG plasmid from competent cells was extracted by using the Qiaprep®Spin Miniprep Kit (QIAGEN GmbH, Germany).
?. [Cloning of reporter luciferase gene under PfSir2A promoter]
The amount of extracted plasmid vector was determined using NanoDrop spectrophotometer 1000 (Thermo Scientific, USA). This was used to calculate the quantity of enzymes needed to linearize the vector.
?. [Cloning of reporter luciferase gene under PfSir2A promoter]
A large quantity of the extracted plasmid was digested in a 100 µl reaction, composed of the ingredients outlined in the table below, at  for 2 h. ComponentQuantity (µl)10X Conc. Yellow  Buffer 20pLNSG (46.3 ng/µl)43.2Enzymes0.5 of eachddH20up to 100
37 °C
ComponentQuantity (µl)10X Conc. Yellow  Buffer 20pLNSG (46.3 ng/µl)43.2Enzymes0.5 of eachddH20up to 100
?. [Cloning of reporter luciferase gene under PfSir2A promoter]
The digested pLNSG was purified using agarose gel electrophoresis (0.8 percent agarose in 1X TBE, which was made from 5X TBE) and the level of purity/intactness assessed visually.
?. [Cloning of reporter luciferase gene under PfSir2A promoter]
The backbone (pLNSir2apro) was extracted from the gel after by using the QIAquick®Gel Extraction kit (QIAGEN GmbH, Germany).
?. [Cloning of reporter luciferase gene under PfSir2A promoter]
The extract from 1.10 was digested a second time using half of the original concentrations of enzyme and 0.5 µl of alkaline phosphatase (1 U/µL, Thermo scientific, USA) was added 30 min before the end of the digestion to reduce the chance of self-ligation.
?. [Cloning of reporter luciferase gene under PfSir2A promoter]
The double digested plasmid was then purified by phenol chloroform clean up and ethanol precipitation. Phenol chloroform clean up involved adding equal volume of phenol/chloroform/isoamylalcohol (Thermo Scientific, USA) solvent mixture and vortexing to extract all the enzymes and other contaminating protein/additives. This mixture was separated into an aqueous and non-aqueous layer by centrifugation at 8000 rpm for 1 min. The aqueous layer was collected using a pipette and put into fresh 1.5 ml eppendorf tube.
?. [Cloning of reporter luciferase gene under PfSir2A promoter]
For the ethanol precipitation, 0.1 volume of 3 M sodium acetate was then added to the aqueous layer and mixed. Thereafter, 2 volumes (2X volume of the above mixture) of absolute ethanol was added to the above, mixed and left at , overnight or at , for 2 h so as to de-salt and precipitate the DNA out from the solution.
-20 °C
-80 °C
?. [Cloning of reporter luciferase gene under PfSir2A promoter]
Afterwards, the mixture was centrifuged at  , 14000 rpm, for 30 min. Ensure that the centrifuge is chilled before use.
4 °C
?. [Cloning of reporter luciferase gene under PfSir2A promoter]
The supernatant was almost completely removed and the pellet formed was washed with ice cold 70% ethanol by spinning at 4°C, 14000 rpm, for 10 min.
?. [Cloning of reporter luciferase gene under PfSir2A promoter]
Purification and amplification of the Fluc insert : The Fluc or simply luc (abbreviation for luciferase gene) coding sequence was amplified from the template pmPLP1, a generous gift from P. Horrocks laboratory, Keele University using polymerase chain reaction (PCR) and the following luc primers; GATCCCTAGGATGCATGAAGACGCCAAAAA (LUCAvrII Forward) and GATCCTTAAGTTACAATTTGGACTTTCCGCC (LUCAflII Reverse).
?. [Cloning of reporter luciferase gene under PfSir2A promoter]
The primers were designed using the SnapGene software and the oligonucleotide sequences were ordered from Eurofins mwg®. The primers had the AvrII and AflII enzyme site attached at the ends.
?. [Cloning of reporter luciferase gene under PfSir2A promoter]
PCR was carried in a reaction mixture containing the components are in the table below:  Component    Quantity (µl)    5X MyFi Buffer (Bioline, USA)    10    Template (1 ng/µl)    2.5     Primers (10 µM)    1 (0.5 each) (0.1 µM final
  concentration of each primer)    MyFi DNA Polymerase (Bioline,
  UK)    2    ddH2O    Up to 50  The PCR conditions were:
Component    Quantity (µl)    5X MyFi Buffer (Bioline, USA)    10    Template (1 ng/µl)    2.5     Primers (10 µM)    1 (0.5 each) (0.1 µM final
  concentration of each primer)    MyFi DNA Polymerase (Bioline,
  UK)    2    ddH2O    Up to 50
?. [Cloning of reporter luciferase gene under PfSir2A promoter]
The PCR product insert was purified by agarose gel electrophoresis, extracted from the gel using the QIAquick®Gel Extraction kit (QIAGEN GmbH, Germany) and restriction digested with AflII and AvrII.
?. [Cloning of reporter luciferase gene under PfSir2A promoter]
The digest was then purified by phenol chloroform clean up and ethanol precipitation. 1 µl of the cleaned up PCR product and plasmid vector backbone were analysed by using agarose gel electrophoresis and the NanoDrop spectrophotometer.
?. [Cloning of reporter luciferase gene under PfSir2A promoter]
Ligation of the backbone and the insert, and detection of positive clones
?. [Cloning of reporter luciferase gene under PfSir2A promoter]
The luc insert and linearized plasmid vector were ligated using a T4 ligase (Thermoscientific, USA) in a thermocycler at , overnight. The formula used for this reaction is shown in the table below. A control reaction was included to determine the extent of self-ligation or re-cloning of uncut vectors.   Component    Test (µ<b style="mso-bidi-font-weight:
  normal">l)    Control (µ<b style="mso-bidi-font-weight:
  normal">l)    10X   ligase buffer (Thermo Scientific, USA)    2     2    Plasmid
  pLNSir2apro backbone (18.75 µg/ml)    0.8   (15 ng)    0.8  (15 ng)    T4 ligase (5 U/µl)    0.5   (2.5 units)    0.5   (2.5 units)    luc PCR product insert (18.75 µg/ml)    8&nbsp; (150 ng)    &nbsp; -    ddH2O    Up   to 20    Up   to 20
16 °C
Component    Test (µ<b style="mso-bidi-font-weight:
  normal">l)    Control (µ<b style="mso-bidi-font-weight:
  normal">l)    10X   ligase buffer (Thermo Scientific, USA)    2     2    Plasmid
  pLNSir2apro backbone (18.75 µg/ml)    0.8   (15 ng)    0.8  (15 ng)    T4 ligase (5 U/µl)    0.5   (2.5 units)    0.5   (2.5 units)    luc PCR product insert (18.75 µg/ml)    8&nbsp; (150 ng)    &nbsp; -    ddH2O    Up   to 20    Up   to 20
?. [Cloning of reporter luciferase gene under PfSir2A promoter]
The resulting pLNSir2aproluc (PfSir2A-luc reporter) construct was purified by phenol chloroform clean up and precipitated using the ethanol precipitation method  - . 1 µl of yeast tRNA (Thermoscientific, USA) was added to a final concentration of 0.2 µg/µl, before the sodium acetate was added during the clean-up of the construct.
?. [Cloning of reporter luciferase gene under PfSir2A promoter]
The construct was dissolved in 10 µl of ddH20 and 5 µl of the construct solution was then used to transform 50 µl of electrocompetent E. coli by electroporation as described  - . The recovered culture was centrifuged at 6000 rpm for 1 min to form a pellet.
?. [Cloning of reporter luciferase gene under PfSir2A promoter]
200 µl of the resulting clear supernatant were discarded and the bacterial pellets were gently mixed with the remaining supernatant and cultured by spreading on an oven-dried sterile LB agar plate containing 100 µg/µl of ampicillin. This plate was incubated at , overnight.
37 °C
?. [Cloning of reporter luciferase gene under PfSir2A promoter]
The ratio of colonies on the test plate (luc:: pLNSir2apro ligation) to the control plate (pLNSir2apro alone) was checked, then a number of colonies from the test plate was subcultured separately in 2 ml LB broth, containing 100 µg/µl of ampicillin at 37°C, overnight, in a shaking incubator.
?. [Cloning of reporter luciferase gene under PfSir2A promoter]
The plasmid was extracted from the resulting broth culture of each colony using the Qiaprep®Spin Miniprep Kit (QIAGEN GmbH, Germany) and digested using AvrII and AflII. The digest was run on the gel to identify positive clones.
?. [Cloning of reporter luciferase gene under PfSir2A promoter]
Plasmids from two positive clones were sent for sequencing by Eurofins mwg® using 4 sequencing primers; GATCCCTAGGATGCATGAAGACGCCAAAAA (LUCAvrII Forward), GATCCTTAAGTTACAATTTGGACTTTCCGCC (LUCAflII Reverse), GGGGTGATGATAAAATGAAAG (hsp86term_Seq) and GCAATTGTTCCAGGAACCAG (LUC_Seq) generated in Snapgene software (GSL Biotech LLC). The sequence trace was then aligned with the original sequence in Snapgene software (GSL Biotech LLC) and any mutation was verified.
?. [Cloning of reporter luciferase gene under PfSir2A promoter]
Preparation of maxi preps for large scale extraction of PfSir2A-luc reporter construct : This protocol is similar to plasmid mini prep but involves the use of supersize kits and equipment needed for preparation of large amounts of plasmids used in transfecting large populations of protozoa or mammalian cells, and for low copy number plasmids.
?. [Cloning of reporter luciferase gene under PfSir2A promoter]
E. coli carrying the PfSir2a-luc reporter construct was grown in 500 ml LB broth, overnight in a shaking incubator at 37°C.
?. [Cloning of reporter luciferase gene under PfSir2A promoter]
The plasmid was then extracted from the resulting culture using the large scale QIAGEN Plasmid Maxi Kit (QIAGEN GmbH, Germany) with a yield of 578 µg.  Digestion of a small aliquot of the extracted plasmid construct was used to validate the plasmid.
?. [Cloning of reporter luciferase gene under PfSir2A promoter]
The plasmid was precipitated by ethanol precipitation and 50 µg was dissolved in 30 µl of ddH20. This was then used for the subsequent transfection of P. falciparum 3D7.
?. [Cloning of reporter luciferase gene under PfSir2A promoter]
Complete restriction analysis of the plasmid construct : PfSir2a-luc construct were restricted using 5 restriction enzymes that cleave at various restriction sites in the plasmid which resulted in different sized segments containing the different components of the plasmid.
?. [Cloning of reporter luciferase gene under PfSir2A promoter]
The following FastDigest enzymes (ThermoFisher, UK) were gently thawed and used to restrict the PfSir2A-luc construct: EcoRV, AvrII, ApaI, SacI and SalI. The reaction components and conditions are presented in the table below.  Components/Conditions    Reaction quantity/condition in each digest    Tube 1    Tube 2    Tube 3    Tube 4    Tube 5  Tube 6  10X FastDigest Green Buffer
  (µl)  (ThermoFisher, UK)    1    2    2    3    4    5    PfSir2A-luc construct (µl)    1    2    2    2    2    2    Number of enzymes     -    1    2    3    4    5    ddH20 (µl) up to     10    20    20    30    40    50    Incubation time (min)    20    5    10    13    20    20    Incubation temperature (°C)    37    37    37    37    37    37
Components/Conditions    Reaction quantity/condition in each digest    Tube 1    Tube 2    Tube 3    Tube 4    Tube 5  Tube 6  10X FastDigest Green Buffer
  (µl)  (ThermoFisher, UK)    1    2    2    3    4    5    PfSir2A-luc construct (µl)    1    2    2    2    2    2    Number of enzymes     -    1    2    3    4    5    ddH20 (µl) up to     10    20    20    30    40    50    Incubation time (min)    20    5    10    13    20    20    Incubation temperature (°C)    37    37    37    37    37    37
?. [Transfection of P. falciparum 3D7 with PfSir2A-luc reporter construct and characterization of the transgenic parasite line.]
Transfection of P. falciparum 3D7 with PfSir2a-luc reporter construct : P. falciparum 3D7 was transfected with PfSir2a-luc reporter construct. This was performed as previously described with some modifications.
?. [Transfection of P. falciparum 3D7 with PfSir2A-luc reporter construct and characterization of the transgenic parasite line.]
50 µg of the plasmid was first introduced into uninfected red blood cells (RBCs) that had been washed in incomplete CytoMix, . This introduction was carried out by electroporation of a mixture of 30 µl of a solution of the plasmid construct (1.67 µg/µl) in sterile ddH2O, 170 µl of CytoMix and 200 µl of the washed RBCs, in a 0.2 cm cuvette  at 0.31 kV and at 960 or 950 µF (microfarads), aiming for a time constant of between 10 to 14 ms.
?. [Transfection of P. falciparum 3D7 with PfSir2A-luc reporter construct and characterization of the transgenic parasite line.]
The DNA loaded RBCs were then recovered with 5 ml of complete media and washed twice with the complete media in a Falcon tube.
?. [Transfection of P. falciparum 3D7 with PfSir2A-luc reporter construct and characterization of the transgenic parasite line.]
0.5 ml of the parasites in untransformed RBCs at about 5% parasitaemia was then cultured in 4.5 ml of the transformed RBCs in complete media at ~2.5% haematocrit.
?. [Transfection of P. falciparum 3D7 with PfSir2A-luc reporter construct and characterization of the transgenic parasite line.]
They were allowed to multiply, reinvade the transformed RBCs, and spontaneously take up the introduced plasmid. 2 µg/ml (of the final culture volume) of Blasticidin-S deaminase (BSD), a drug that selects for the transfected parasites, was then added 48 h later.
?. [Transfection of P. falciparum 3D7 with PfSir2A-luc reporter construct and characterization of the transgenic parasite line.]
The parasite culture media was regularly changed in order to remove dead parasites and refresh the parasite nutrients. The drug was added with each media change until drug resistant parasites emerged and thereafter.
?. [Transfection of P. falciparum 3D7 with PfSir2A-luc reporter construct and characterization of the transgenic parasite line.]
50 µl of 100% RBCs was added every week until the transfected parasites came up. Parasite detection was done by microscopy and bioluminescence signal was measured.
?. [Transfection of P. falciparum 3D7 with PfSir2A-luc reporter construct and characterization of the transgenic parasite line.]
Detection of transgenic parasites : The protocol used to detect this luciferase activity of parasite culture was the improved Single-Step Lysis Protocol . This was done using a luciferase assay system (Promega, UK) and a luminometer, Glomax-Multi Detection System (Promega, UK). The luciferase assay system contained 5X Passive lysis buffer (PLB) and Luciferase assay substrate (LAS).
?. [Transfection of P. falciparum 3D7 with PfSir2A-luc reporter construct and characterization of the transgenic parasite line.]
In triplicate well per sample, 10 µl of 5X PLB, warmed to 37°C, was homogenized with 40 µl of parasite culture in each of the wells of a 96-multiwell white plate (Grenier-bio-one, Germany), by rocking/swirling.
?. [Transfection of P. falciparum 3D7 with PfSir2A-luc reporter construct and characterization of the transgenic parasite line.]
50 µl of LAS was then added to the mixture in each well at room temperature and mixed by rocking/swirling.
?. [Transfection of P. falciparum 3D7 with PfSir2A-luc reporter construct and characterization of the transgenic parasite line.]
The bioluminescence was measured in light unit using the Glomax-Multi detection immediately.
?. [Transfection of P. falciparum 3D7 with PfSir2A-luc reporter construct and characterization of the transgenic parasite line.]
Characterization of 3D7 PfSir2A-luc reporter line
?. [Transfection of P. falciparum 3D7 with PfSir2A-luc reporter construct and characterization of the transgenic parasite line.]
Additionally, tightly synchronized parasites were used to determine the bioluminescence of the 3D7 PfSir2A luciferase reporter line, alongside the well characterized Dd2 luciferase, Pfpcna-luc, reporter line throughout their IDC. The experiment was begun 55 h and 48 h after the Percoll and sorbitol synchronizations respectively (i.e. 7 h post invasion (hpi)) and the parasites conformed to the required stage as assessed by Giemsa stained thin blood smear which indicated at least 82% purity of the required stage.
?. [Transfection of P. falciparum 3D7 with PfSir2A-luc reporter construct and characterization of the transgenic parasite line.]
The parasite samples were harvested when they were at most 7, 10, 21, 24, 27, 30, 45, 48, 51, and 54 hpi and their bioluminescence determined . The signal was normalized to 1% parasitaemia.
?. [Transfection of P. falciparum 3D7 with PfSir2A-luc reporter construct and characterization of the transgenic parasite line.]
A thin blood smear was also made for the different time points, stained and photographed using Leica® camera attached to a light microscope.
?. [Transfection of P. falciparum 3D7 with PfSir2A-luc reporter construct and characterization of the transgenic parasite line.]
Asynchronous parasite population of 3D7 PfSir2A-luc reporter line was used to determine the effect of parasitaemia on bioluminescence by serial dilution from 2.5% parasitaemia down to 0.5% parasitaemia.
Constantly mix the parasite culture while diluting so as to ensure even distribution of the parasites.