1. BS 592 Project Stage II
Heterologous expression & Purification of Plasmodium falciparum TGS1 in E. coli
Project Report Stage II
Submitted by
Soumya Ranjan Sahu
Roll.No.12i300007
In the partial fulfillment of the requirements for the award of the degree of
Master of Science (Biotechnology)
Guide: Prof. Swati Patankar
Department of Bioscience and Bioengineering
Indian Institute of Technology Bombay
Bombay 400076
April 2014

2. Letter of consent
The work reported in this dissertation entitled "Heterologous expression &
Purification of Plasmodium falciparum TGS1 in E. coli"has been carried out by Soumya
Ranjan Sahu (12i300007) under my guidance in my laboratory. I hereby approve the
submission of the project report.
Guide: Prof. Swati Patankar
Place:
Date:

3. Plagiarism Undertaking
I , Soumya Ranjan Sahu (Roll No. 12i300007) understand that plagiarism is defined as
any one or the combination of the following:
1. Uncredited verbatim copying of individual sentences, paragraphs or illustrations (such
as graphs, diagrams, etc.).
2. Uncredited improper paraphrasing of pages or paragraphs (by changing a few words or
phrases, or rearranging the original sentence order).
3. Credited verbatim copying of a major portion of a paper (or thesis chapter) without clear
delineation of who did or wrote what.
I have made sure that all the ideas, expressions, graphs, diagrams, etc., that are not a
result of my work, are properly credited. Long phrases or sentences that had to be used
verbatim from published literature have been clearly identified using quotation marks.
I affirm that no portion of my work can be considered as plagiarism and I take full
responsibility if such a complaint occurs. I understand fully well that the other authors of this
paper or guide of the thesis / dissertation may not be in a position to check for the possibility
of such incidences of plagiarism in this body of work.
Signature with Date
Name: Soumya Ranjan Sahu
Roll Number: 12i300007

4. CONTENTS
1. Introduction
1.1 Malaria
1.2 P. falciparum
1.3 Life Cycle Of P. falciparum
1.4 Insertions In P. falciparum Proteins
1.5 SnrnaAnd Splicing
1.6 Tgs1 Of P. falciparum
1.7 Reasons To Study PfTGS1
1.8 Nuclear Localization Signal
2. Objective
3. Strategy
4. Materials And Methods
4.1 Finding The Protein & Gene Sequence Of PfTGS1
4.2 Finding The Functional Domains Present In PfTGS1
4.3 Finding Gene Sequence OfMTD
4.4 Selecting A Vector For Expressing This Protein In Bacteria
4.5 Designing Primers For Amplification OfMTD
4.6 Amplifying MTDWith Proper Restriction Sites
4.7 Isolation Of pET3a Plasmid From E. coli Dh5α By Alkaline Lysis Method
4.8 Restriction Digestion Of Plasmid
4.9 Restriction Digestion OfAmplifiedMTD With Restriction Enzymes
4.10 Purification Of Double Digested Plasmid And Double Digested PfTGS-1 MTD
PCR Product
4.11 Ligation OfDoubleDigeseted Plasmid With Doule Digested MTD

5. 4.12 Making Of Competent Cells For Transformation
4.13 Transformation Of Ligated Product To E.coli Cells
4.14 Colony PCRFor Finding The Colony/Cells With Recombinant Plasmid
4.15 Purification of Protein with Ni NTA column
4.16 Dialysis of the Purified protein
5. Results
5.1 Result Of FindingMTDIn Pftgs1 And Designing Its Primer
5.2 Result OfPlasmid Isolation
5.3 Result Of PCR Amplification Of PfTGS1-MTD
5.4 Calculation For Ligation Reaction
5.5 Result Of Double Digestion Of PfTGS1-MTDAndpET43a
5.6 Reaction Table For Ligation
5.6 Reaction Table For Ligation
5.7 Transformation Efficiency Of The Prepared Competent Cell
5.8 Result Of Transformation Of Ligated Product With Competent Cells
5.9 Result Of Colony PCR
5.10 Result of Ni NTA Purification
6. Discussion
7. References

6. LIST OF ABBREVIATIONS
MTD- Methyltransferase domain
V- Vector
I- Insert
L- Ligase
P. falciparum- Plasmodium falciparum
LA- Luria Agar
LB- Luria Broth
LA+ ampicillin- Luria agar plates with ampicillin
w.r.t.- with respect to
PfTGS1- TGS1 of Plasmodium falciparum
GFP- Green fluorescent protein

7. 1.INTRODUCTION
1.1 MALARIA:
Malaria is a mosquito borne disease caused by protozoan parasites of the genus
Plasmodium. Four species of Plasmodium infects human, viz P. falciparum, P. vivax, P.
malaraiae and P. ovale. In 2010 an estimated 219 million cases of malaria occurred
worldwide and 660,000 people died among which 91% were from the African Region
1.2 P. falciparum :
P. falciparum is the deadliest parasite, causing more than one million malaria-related
deaths per year in endemic areas of sub-Saharan Africa. 90% of the deaths are due to P.
falciparum (Kim, Y. & Schneider).Plasmodium falciparum is a unicellular eukaryote. It is
transmitted by various species of female anopheles mosquito. Parasites reproduce asexually in
human hosts whereas sexual cycle is completed in mosquito. The parasite life cycle has been
shown in Figure 1. Since 2002, the complete genome sequence of P. falciparum is available
(Gardner et al.). This has boosted the bioinformatics studies in this parasite. These
bioinformatics studies showed that Plasmodium falciparum proteins are longer than their
orthologs because of the presence of insertions (Aravind et al., 2003).The A/T content of the
entire genome is more than 80%. The A/T content in the insertions is even much higher than
that of the genome (more than 90%) (Gardner et al.).
To develop a drug against this deadly parasite, it is essential to understand the
biology of the parasite. In some cases it has been observed that the biological processes of the
parasite are different from other model organisms.

8. 1.3 LIFE CYCLE OF P. falciparum
(Image courtesy-http://www.cdc.gov/malaria/)
Figure 1. Showing life cycle of P. falciparum
1.4 INSERTIONS IN P. falciparum PROTEINS
Insertions are extra stretches of amino acid sequences that are absent in its other
homologous sequences. From the homologous series of proteins, the protein with longer
sequence is said to be having insertion in it w.r.t. to the protein with shorter sequence and the
protein with shorter sequence is said to having deletions w.r.t. the protein with longer
sequence. It has been thought that the insertions found in parasite proteins should generally
loop out of the globular structure of the protein. The reason for this is the fact that the
insertions are composed of highly polar amino acids. Insertions may be of high complexity or
low complexity. Low complexity region (LCR) is composed of limited set of amino acids
whereas high complexity region are having larger set of amino acids. Nearly 90% of P.
falciparum proteins contains at least one LCR (Gardener et al.,2002), including many highly
conserved housekeeping genes (Pizzi and Frontali, 2001; Aravind et al., 2003).

9. Insertions are expected to form extended loops and they do not form any globular
structures (Wootton, 1994). So, the insertions do not affect protein function much more. But
there are few examples of functional importance of these insertions are shown. In P.
falciparum subtilisin-like protease-1, insertions form surface exposed loops and their deletion
affects the enzyme stability and activity (Jean et al., 2005).
It has been seen that in P. falciparum protein insertions are generally rich in charged
amino acids thus the hydropathy index of the insertions is low(Pizzi and Frontali, 2001).
Insertions are generally surface exposed due to low hydropathy index.
1.5 snRNA and splicing
Small nuclear RNA is an integral component of the spliceosome which carry out
splicing of the mRNA (Guo et. Al). These are associated with specific set of proteins and
forms complexes called as snRNP often referred as snurps. snRNP forms complex with
unmodified pre mRNA forming spliceosomal complex and then splicing of mRNA takes
place. snRNP particles are composed of several Sm proteins, the snRNA component, and
snRNP specific proteins.
Splicing is a modification of pre mRNA formed after transcription to matured
mRNA by removal of the introns and then rejoining of the exons. All these steps occur in the
nucleus. snRNA is formed in the nucleus by transcription mediated via RNA polymerase II
and receive a 5’ monomethylguanosine cap. This 5’ mono-methylguanosine cap acts as
nuclear export signal for moving it to the cytoplasm. In cytoplasm it is processed and matured.
Trimming of 3’ and hypermethylation of 5’ end occurs. Hypermethylation is supposed to
mediate by enzyme TGS1.
1.6 TGS1 OF P. falciparum
More than 50% of the genes of P. falciparum contain introns, there should be an
efficient mechanism present in P. falciparum. This indicates that an efficient TGS1 should be
present in P. falciparum. A gene encoding the enzyme for hypermethylation of snRNA cap
structures (TGS1) is present in the P. falciparum genome. The snRNA trafficking protein
Snurportin is absent from the P. falciparum genome suggesting that parasite snRNAs and

10. TGS1may be retained in the nucleus (Bawankar et al.). PfTGS1 is larger than its orthologs and
may have transmembrane domains in the C-terminus (bioinformatics analysis). It contains one
insertion. Functions of this insertion are not clearly known. Structure of conserved
Methyltranferase domain of human TGS1 is available.
Insertions are common features of all Plasmodium proteins. It is an important protein
involved in cellular processes. Our laboratory works on the 5’ end modification of RNA. If the
functions and localization of TGS will be known, then it will be easier to design drug
molecules to target its life cycle.
2. OBJECTIVES
1. Clone PfTGS1 methyl transferase domain in pET43a expression vector.
PfTGS1 methyl transferase domain will be PCR amplified using suitable primers
from genomic DNA of P. falciparum. Previously pET28a vector was used for the expression
of this protein but it has been observed that the protein goes into the insoluble fraction. To
overcome this difficulty attempt to express PfTGS1 methyl transferase domain with NUS tag
had been made.
2. Expression of PfTGS1 methyl transferase domain in BL21DE3:
Conditions will be optimized for the expression of the protein in BL21DE3 strain.
3. Purification of the PfTGS1 methyl transferase domain from bacterial lysate using Ni-
NTA beads.
If the protein is expressed successfully in bacteria, purification of the protein will
be optimized using Ni-NTA beads.
3.STRATEGY

11. 1. Cloning of Methyltransferasedomain(MTD) of PfTGS1 in E. coli with pET43a
plasmid (pET43a contains His tag and NUS tag).
2. Expressing the protein in E.Coli by induction with IPTG.
3. Purification of protein with Ni-NTA beads
4.MATERIALS AND METHODS
4.1 Finding The Protein & Gene Sequence Of PfTGS1
PFL0125c (Gene ID of PfTGS1) was searched at PlasmoDB (plasmodb.org) and the
gene and protein sequence are saved.
4.2Finding The Functional Domains Present In PfTGS1
The protein sequence was analyzed at website of SMART (smart.embl-
heidelberg.de).
4.3 Finding Gene Sequence Of MTD
The position of MTD was founded by SMART. Accordingly its genome sequence can
be determined by calculation keeping in mind that one amino acid is coded by 3 nucleotide
bases of the gene. It was checked with reverse translation tool at EXPASY.
4.4 Selecting A Vector For Expressing This Protein In Bacteria
pET43a is chosen to express this protein in bacteria because of some unique properties
of this plasmid. It has T7 lac promoter It contains several restriction sites including Nus Tag
(834-2318). BamHI (660) and Xho I (530) are selected as restriction sites.
4.5 Designing Primers For Apmplication Of PfTGS1-MTD (238-398)
This primer design can be done at NCBI Primer-BLAST putting the coding sequence
in FASTA format. Primer should be of 20-30 nucleotides long with proper GC content for
strong binding. The first few sequences from 5’ end (20-30 nucleotides) of the coding
sequences is selected as forward primer (Sites?).For designing reverse primer, reverse

12. complement of the last 20-30 nucleotide sequences was taken.Restriction site sequences are
added to both primers. BamH I is sequenced to the 5’ end of the forward primer and Xho I at
the 5’ end of the reverse primer. Four random nucleotides are added further to the 5’ ends of
the primer.
4.6 Amplifying PfTGS1-MTDWith designed primers
Designed primers were obtained from the manufacturers (Sigma) PCR was
optimized looking at the melting and annealing temperature of the primers. The buffer
concentration and Mg++
ion concentration were also optimized to get maximum PCR products
of our interest. The optimized PCR reaction is-
For 25 µl of PCR reaction mixture, used components are-
5 µlof 5X kapahifi buffer + 1.5 µl of 25 mM MgCl2+ 0.5 µl of forward primer (10
pmol/µl) + 0.5 µl of reverse primer (10 pmol/ µl) + 0.5 µl of 10mM dNTP (mixture) + 2 µl of
genomic DNA(P. falciparum) +14µl of distilled water
4.7 Isolation OfpET43a Plasmid From E. coli DH5α by Alkaline Lysis Method
E. coli containing this plasmid was picked up from the LA+ ampicillin plate where
it was grown previously and was inoculated in 5ml LB medium containing ampicillin
(100µg/ml). It was kept on a shaker for 12-16 hours at 37o
C. 1.5 ml of culture was transferred
to an eppendorf and centrifuged at 10000g for 2 minutes. The supernatant was discarded
and again 1.5 ml culture was added to the same eppendorf and the process was repeated. The
bacterial pellet was resuspended in100µl of solution I [50mM glucose, 25mM Tris-HCl (pH
8.0), 10mM EDTA(pH 8.0)] by vigorous vortexing. To this added 0.2 ml of solution II [0.2N
NaOH and 1% SDS (w/v)]`which is freshly prepared and mixed gently by inverting he 5-6
times and incubated on ice for next 5 minutes. 1.5ml of solution III [3M CH3COOK AND 5M
glacial acetic acid] was added to viscous bacterial lysate. Gently mixed by inverting 4-5 times
and incubated on ice for next 5 minutes. The tube was centrifuged at 12000g for 5 minutes.

13. And the supernatant was transferred to another fresh tube. An equal volume of
phenol:chloroform:isoamylalcohol (25:24:1 by volume) was added to the supernatant and
centrifuged at 12000g for 5 minutes at 4o
C.The upper aqueous layer was removed carefully
and transferred to another eppendorf. Double volume of 100% ethyl alcohol was added to it
and kept at RT for 20 minutes. Then the tube was centrifuged at 12000g for 15 minutes at 4o
C. The supernatant was discarded and the pellet was washed with ethanol(70%) and the tube
was kept at 45o
C for 30 minutes. The DNA pellet was dissolved in 40µl of deionized distilled
water.
4. 8 Restriction Digestion of Plasmid
The plasmids were double digested with restriction enzymes BamH I and Xho I.
For this 10X Tango buffer is used. For a 20µl reaction tube 6 µl of plasmid vector, 0.5 µl each
of the enzymes,0.5 µl of fast alkaline phosphatase and 0.2 µl of RNase was added and rest
make up with sterile distilled water. All the components were mixed and kept at 37o
C for 3
hours.
Tango buffer is designed for double digestion of DNA with conventional restriction
enzymes. It contains BSA, which enhances the stability of many enzymes and bind to the
contaminants that may be present in DNA preparations for consistent enzyme performance. It
is composed of Tris acetate, magnesium acetate, potassium acetate and BSA.
4.9 Restriction Digestion Of Amplified MTD With Restriction Enzymes
The amplified MTD of PfTGS1 contains restriction sites for BamH I and
XhoIwhich were to be digested with proper enzymes before ligation of vector and insert. Sam
eprotocol is followed here as it was done for double digestion of plasmidexpect that RNase
and fast alkaline phosphatase were not used here.
4.10 Purification Of Double Digested Plasmid And Double DigestedPfTGS1-MTD PCR

14. For purification of double digested plasmid and double digested PfTGS1-
MTD,Thermo Scientific, GeneJET Gel Extraction Kit (#K0691, #K0692) is used with slight
modification of their provided protocol.
X µl of DNA(plasmid/MTD PCR product) was taken. To this 3X µl of Binding
buffer was added. To this 2.5X µl of isopropanol a added. Then the solution was added to
GeneJET purification column and centrifuged at 12000g for 1 minute. The flow through was
again put into the column and process repeated. The flow through was discarded and 7010µl of
wash buffer was added to the column. .It was then centrifuged for next 1 minute at 12000g.
The flow through was discarded and the empty column was again centrifuged for 1 minutes at
12000g. The column was then transferred to a new eppendorf. Then, 30µl of slightly hot sterile
distilled water was added to the center of the column ver carefully. This was centrifuged for 1
minute at 12000g and the flow through was stored at -20 degree C. This was the purified
DNA(plasmid/PCR product).
4.11 Ligation Of Double Digested Plasmid With Double Digested MTD
For this, two controls are set up along with test.
In control 1, there are all components of ligation mixture except insert and ligase(i.e.,
Vector only). From this control,it is possible to quantify that how many colonies are formed
due to transformation of parental uncut plasmid on the LA+ ampicillin plates after
transformation. This will also provide an idea of the efficiency of the restriction enzyme.
In control 2, there are all components of the ligation mixture except the insert
(vector+ligase). From this ,it will be easier to quantify, how many colonies are there on the
LA+ ampicillin plates due to transformation of self ligated plasmid after transformation. This
will also provide an idea about efficiency of the fast alkaline phosphatase.
In test,there were all components of the ligation mixture along with double digested
vector(pET43a),double digested insert (MTD PCR product) and ligase enzyme were
present.For 20 µl ligation reaction mixture, 10X ligase buffer is used along with10mM ATP,
Vector(154 ng), Inse rt(44 ng) ,Ligase enzyme and rest up the volume by sterile distilled
water.

15. 4.12Making Of Competent Cells For Transformation
For preparation of competent cells a single colony of E. coli DH5α was inoculated
in 5 ml LB and grown over night in a shaker at 37 degree C. From this 2 ml of the O/N grown
culturewas inoculated in 200 ml LB allowed for growing till 0.6 OD. When it was reached the
desired OD,then it was incubated on ice for next 15-30 minutes. Then the culture were divided
into four falcons(50 ml each). The falcon tubes were centrifuged at 5000g for 8 minutes at 4
degree C. The pellet was re suspended in 10 ml chilled 100 mMCaCl2for each falcon tube. It
was incubated on ice for next 15 minutes. The falcons were then centrifuged at 5000 g for 8
minutes at 4 degree C. Then 1 ml of 1oomMCaCl2was added to the pellets. Then added 0.5 ml
of chilled glycerol (50 %) and mixed gently. Then these were kept as 200 µl in eppendorfs and
kept at -80 degree C.
4.13 Transformation Of Ligated Product To E.coliCells
The competent cell were moved out from -80 degree C and kept on ice for 30
minutes. To this 1 µl of ligated products were added along with controls in separate
eppendorfs. The mixture was incubated on ice for next 30 minutes. Then the tubes were
transferred to 42 degree C and kept for90 seconds. Then the tubes were removed and kept
again on ice for next 5 minutes. Then 1 ml LB was added to the tubes and wasincubated on a
shaker at 37 degree C for next 45 minutes. Then 200 µl from the tubes were taken and
spreaded on LA+ ampicillin plates. Then the plates were kept at 37 degree C for next 16 hours.
Then the plates were removed from 37 degree C, transformation efficiency was calculated and
the plates were kept at 4 degree C.
4.14 Colony PCRFor Finding The Colony/Cells With Recombinant Plasmid
Colony PCR used here is exactly followed the same protocol as described above
except that instead of genomic DNA, a colony was provided as template. Also, Taq
polymerase and Taq buffer (10X) was used instead of kappa hifi enzyme and kappa hi fi
buffer.
For this, 16 colonies are randomly chosen from the Vector + Ligase + Insert Plate and
grown on LA + ampicillin plates by streaking. Then it was kept at 37 degree C for next 16

16. hours. From these streaks of growth, cells were taken as template for PCR and PCR was done
with same primers as used above.
4.15 Purification of Protein with Ni NTA column
Transformed cells with recombinant plasmid were cultured in LB media and kept in
-800
C in glycerol stocks . LB tubes with 100 µg/ml of ampicillin were inoculated with culture
containing the gene for MTD. These were grown overnight in shaking condition of 200rpm at
37 degree C. 5 ml of this culture were inoculated in 500 ml LB containing ampicillin. This was
again kept in same shaking condition till it reaches OD of 5-5.5 . After that IPTG is added to it
and kept in same shaking condition for next four hours. Concentration of IPTG was already
optimized before this by inducing with different concentrations of IPTG
(0.2mM.0.5mM,0.8mM & 1.0mM). After this , the culture were divided into two 250 ml
tubes for centrifugation. Then centrifugation was done with 7000 rpm for 15 minutes at 4
degree C. Pellets kept at -80 degree C. The pellet were brought out of -80 degree C and kept
on ice. 5-6 ml of lysis buffer were added to each tube containing pellet and mixed thoroughly
by gently reverse pipetting. Then solution from both the tubes are transferred to a 50 ml falcon
tube and again kept on ice.. Then this solution was sonicated on ice with 60 % amplitude with
1 second pulse for 30 seconds for nearly 15 times with 1 minute gap between each cycle to
prevent overheating of the sample. Then the sample was centrifuged at 10000g for 40 minutes
and supernatant was collected very carefully without disturbing the pellet. The supernatant was
then incubated with 4 ml Ni NTA beads (50% slurry) for nearly about 5-6 hours on ice and
shaker. Previously the beads were thoroughly washed with distilled water and equilibrated
with binding buffer. Then the solution of Ni NTA beads and supernatant was put on the
purification column and allowed the flow through to pass out. Some of the flow through was
also collected for checking the efficiency of binding. Then the beads were washed three times
with wash buffer 1, two times with wash buffer 2 and with nearly 20 ml with wash buffer 3.
(Composition of wash buffer 1,2&3, lysis/binding buffer & elution buffer are given below for
reference). After that , it is then eluted with nearly 20 ml of elution buffer.
Wash Buffer 1- 50 mM NaH2PO4, 400 mMNaCl& 10 mM Imidazole in D/W
Wash Buffer 2- 50 mM NaH2PO4, 300 mM NaCl& 20 mM Imidazole in D/W
Wash Buffer 3- 50 mM NaH2PO4, 300 mM NaCl& 40 mM Imidazole in D/W

17. Binding/Lysis Buffer - 50 mM NaH2PO4, 300 mMnNaCl& 10 mM Imidazole in D/W
Dialysis Buffer- 6.8 g NaH2PO4 &7.5 mM NaCl in 1 litre D/W (pH7.5)
These eluted volumes are numbered and run through 12 % SDS PAGE for detection of
required protein band.
4.16 Dialysis of the Purified protein
Dialysis Buffer- 6.8 g NaH2PO4 &7.5 mMNaCl in 1 litre D/W (pH7.5).
Dialyis was done for the purified protein to remove the salts and small molecules present in it.
The eluted volumes which show the appropriate protein bands are kept for dialysis by putting
these in a sealed dialysis membrane which are kept in the dialysis buffer with magnetic bead
on a stirrer. Dialysis buffer were kept on changing within every 5-6 hour interval for 3 times.
5.RESULTS
5.1 Result Of Finding MTD In PfTGS1 And Designing Its Primer
>PF3D7_1202500 | Plasmodium falciparum 3D7 | trimethylguanosine synthase, putative |
protein | length=1079
MLKVRYENFNDLRNDDEMLNISFVAHPCYYSILDLEESELFNKIVYENFCKVAEKVDIYD
KNEYLSVYRRHDKKCRELHMLKSWELLKNTYRYKMPVIFKNLKDFDLYNMEKIPFYSINY
NKKKKGTKENGFVVRNKKKINIIDKGKCFILDNHMIYSMTPEYIANNISKNILLNTHHIM
KGNNILSLKEDDKYVKHQKVNDNPYYFKLRYHYNDDDIYCKDNYSYKRKGISPIKKKRKI
EPILYNNKKMDYEEKKRILIYLDPFAGAGGNCNHMNNIFTIGCDINFYRIKQCQHNCNFY
NKNVDFILCDFFNLVTHFRKGTIDVIFFSIPWGGPKYKNKKNFELNTEIMDNISIYKCIE
VSVELTENLVFYLPRNVCMKELYYLYGYYKELVKNKKCVNYENDDIYKYDRKGEGGKKKK
KKKKKKDIKLTSNDTYYYEHKNKLQNRSNNILLELYINRSRCNYQKKDDNSLNNFFYFFN
EKCDIYDNKFIWSKVHNLFNINIDHCYDIFNCGVREKRSFEGEIYNIKEEHVEGESLRYC
IYNRNHENIETCDNKIHSSYDYSYVQDEKKEIKEKGEYSINYSYSNDGNINYPSNRNKKK
NNNKNKKNKIKNKKNIWAWHNTCMVLYLGNISTNIRNKKIINMKDVYYIDKTLSNILIDI
EIGKSKYENFFYSYHNLFFGEKKKRFYCIYKNKKKSFNNKLYINKNELIEKYLINEENIL
YINIFIMKKLYHIIEKIILLFFNNLKNMMGYEKISFLNKICIHLENIYDKILTENIDKHI
DMKKYETYIYKDDEKKGQLFYKHLVEYFNIIIRKIKKEFILLFSIYLYDISFLRYYFRHK
NINIEKKKKKLSTQGLKKIHYNIIRILFNLLLYMKIYMNIVTNNIKLEPYFNQQFIIDDM
NIDHIHKNIINRCSSIYSLRKKKNRNYNFRRNNITSYVSCFKNFIKYVIRICINLELKKN
NEEERISMKNMSNFFFRSLFNVERVDILINKLNNNMNNNEEQEDIKYYKYNLYLLLIHFF
TKQFLYNSHVNTNLDYFNTILFFYFNQIYNLSKYQDGYINIFVHFLNNFLYKNFFLKIT
The predicted domains (by SMART) are shown below

18. The domain within your query sequence starts at position 258 and ends at position 482
ILIYLDPFAGAGGNCNHMNNIFTIGCDINFYRIKQCQHNCNFYNKNVDFILCDFFNLVTH
FRKGTIDVIFFSIPWGGPKYKNKKNFELNTEIMDNISIYKCIEVSVELTENLVFYLPRNV
CMKELYYLYGYYKELVKNKKCVNYENDDIYKYDRKGEGGKKKKKKKKKKDIKLTSNDTYY
YEHKNKLQNRSNNILLELYINRSRCNYQKKDDNSLNNFFYFFNEK
The coding sequence of this MTD is
ATTTTAATTTATTTAGACCCGTTTGCTGGTGCAGGAGGGAATTGTAACCACATGAATAATATATTTACAATAGGTTGTGATATAAATTTTTATAG
AATAAAACAATGTCAACATAATTGTAATTTTTATAATAAAAATGTTGATTTTATTTTGTGTGATTTCTTTAACCTTGTTACCCATTTTAGAAAAG
GCACAATCGACGTTATATTTTTTAGTATACCTTGGGGAGGACCAAAGTATAAGAATAAAAAGAATTTTGAGCTGAACACTGAAATAATGGATAAT
ATAAGTATATATAAATGTATAGAAGTATCCGTAGAACTAACGGAAAATTTGGTTTTCTATCTTCCACGAAATGTATGTATGAAGGAATTATATTA
TTTGTATGGATATTACAAAGAATTAGTAAAGAATAAAAAATGTGTAAATTATGAAAATGATGATATTTATAAATATGATAGAAAAGGGGAGGGGG
GAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGATATTAAGTTGACATCTAACGATACATATTATTATGAACATAAGAATAAGTTGCAAAATAGA
AGTAATAACATATTATTAGAATTATATATAAATCGAAGTAGATGTAATTATCAAAAGAAGGATGATAATAGTTTAAATAATTTTTTTTATTTTTT
TAATGAAAAA
The sequence of forward primer PfTGS1-MTD-F
ATGCGGATCCATTTTAATTTATTTAGACCCGTTTG
The sequence of Reverse primer PfTGS1-MTD-R
TGCACTCGAGTTTTTCATTAAAAAAATAAA

19. 5.2 Result Of Plasmid Isolation
Figure2.showingagarose gel analysis of isolated pET43a with 1 kb ladder
The size of pET43a is 7.3 kb. A RNA band is found in the gel because RNAse treatment
was not followed during plasmid isolation.
5.3 Result OfPcr Amplification Of PfTGS1-MTD
PCR was repeated several times by changing the protocol and finally got optimized to get
maximum amount of MTD amplification for ligation. The size of the expected PCR product
should be of 687bp.

20. Figure 3. Showing PCR amplification of PfTGS1-MTD
The PCR product was purified by GeneJET purification kit (#K0691,thermo scientific).
Figure 4.Agarose gel electrophoresis, PfTGS1-MTD PCR product purified
A very faint band of non specific PCR product was also found.
5.4 Calculation For Ligation Reaction
Size of vector ~7 kb
Size of insert ~ 0.65 kb
For ligation reaction , the ratio of V: I should be 1:3
For 200 ng reaction mixture, 7x×1+0.65x =200
Hence, x=22.35
So, amount of vector needed = 7×1×22.35 = 156.45 ng
Amount of insert required = 0.65×3×22.35 =43.58 ng

21. 5.5 Result Of Double Digestion OfPfTGS1 MTD and pET43a
Figure 5.Agarose gel electrophoresis, purified double digested plasmid with PCR product
of PfTGS1-MTD.
For 200 ng of ligation reaction mixture,the amount of vector and insert required is already
calculated above.
From the above gel pic, it was impossible to calculate the concentration of concenctration
of plasmid and insert. But, there is ~100 ng of PCR product as it is known than minimum
amount of DNA required to visualize on a gel.
So, rough calculation was done to get more molar ratio of I:V .
5.6 Reaction Table For Ligation
Vector
only(µl)
Vector+Ligase
(µl)
Vecor+Insert+Ligase
(µl)
DistilledWater 13.4 10 0
Ligase
buffer(10X)
2 2 2
Vector 3.6 6 6
Insert 0 0 10
Ligase 0 1 1
ATP 1 1 1

22. 5.7 Transformation Efficiency Of The Prepared Competent Cell
The transformation efficiency of the competent cells were checked prior to the original
transformation with recombinant PftTGS1-MTD and it was found to be 105
CFU/µg
5.8 Result Of Transformation Of Ligated Product With Competent Cells
The growth of transformants on LA+ampiciliin plates are as follows:
Number of Colonies
Vector only 23
Vector + Ligase 16
Vector + Insert + Ligase 50
For the vector only, to the ligation mixture tube 6 µl of vector was added and it after
transformation 23 colonies were found on the plate means there were still some uncut pET43a
exist even after double digestion. This number shows the inefficiency of the restriction
enzymes or double digestion procedure.
For Vector + Ligase,3.6 µl of vector were added, and after transformation, 16 colonies
were found on the LA + ampicillin plates. So, by calculations it can be said that, 14 colonies
are due to uncut vector and 2 colonies wre due to cut vector(not affected by fast alkaline
phosphatase) and ligase action.
For Vector + Insert + Ligase, 6 µl of vector was added to ligation mixture tube, and
after transformation, 50 colonies were found. By calculations and comparing with the above
data (from Vector only and Vector + Ligase), it can be said that, 23 colonies were due to uncut
vector, 4 colonies are due to cut vector ( un affected by fast alkaline phosphatase) and ligase
and rest 23 colonies are expected to be perfect recombinants.
5.9 Result Of Colony PCR
All the 16 PCR tubes are analyzed by agarose gel electrophoresis with positive
control (all components for PCR with P. falciparum genomic DNA as template) and negative
control (all components of PCR except any template).

23. Figure 5.Agarose gel electrophoresis analysis of colony PCR samples
( Figure 6. Agarose gel electrophoresis analysis of colony PCR samples)
From the analysis of the randomly picked 16 colonies, none of the colonies seems to have
the recombinants as no PCR amplifications was found. There should be some PCR product of
same size if the cells have the recombinant plasmid.
5.10 Result Of Ni NTA Purification
For checking and optimizing induction, first the cells were induced with different
concentrations of IPTG and checked with uninduced ones.

24. (Figure 7.SDS PAGE run after induction with different IPTG conc.)
From the above gel picture, it can be clearly seen that there is a good amount of
induction with 0.5 mM of IPTG. So for all further induction of this protein, 0.5mM of IPTG
was used. The size of the protein PfTGS1 –MTD with NUS tag is calculated to be appx 84 kD
and here from induction it shows to be appearing slightly lower then the 91 kD ladder.
Below is the gel picture after purification through Ni NTA column. The protein was
found to be present in elution 3 & 4. The band showing just below 91 is the expected protein.
Column 3 & 4 are elution 3 & 4 respectively.
(Figure 8. SDS PAGE of the protein after purification with Ni NTA)
Below is the gel picture of the purified protein after dialysis.
(Figure 9. SDS PAGE after dialysis)
After dialysis , it seems to be the protein band of our interest is of very low concentration.
Although PMSF was used in the dialysis buffer, the reason of the dissaperance of the protein is
still unknown.

25. 6. DISCUSSION
Colony PCR indicates that there are no recombinants on the plate. The ratio of number
of colonies on Vector only : Vector +Ligase+ Insert is 23:50 (~1;2). This ratio is not optimum
to expect the presence of true recombinants. Also the transformation efficiency of the prepared
competent cells is of the order of 8X105
which is lower than the competent cells generally
used in the lab. Our lab has now developed a new protocol for efficient competent cell
preparation. So, hence onwards, these competent cells will be used for doing
transformation.Also, the yield of plasmid decreases after clean up. This problem can be
surmised using higher amount of plasmid in the digestion reaction itself.
From the Colony PCR done by me, it was clear that there were no recombinants in any
of the transformed cells. Once again transformation was tried in our lab and was successful
having recombinant BL21 DE3 strain . From these cells the PfTGS1-MTD was induced
protein is purified.
The exact size of the protein was found to disappeared after dialysis and . Protein
purification was also tried two more times but eluted with a very low amount and also
disappeared after dialysis even though there is proper induction. The low amount of protein
after Ni NTA purification may be due to improper binding of the protein with the Ni NTA
beads. So, the binding efficiency of the beads should be checked with other known His tagged
protein . Also western blot with antiHis antibody to be done for checking that His tag is
available in our target protein.
After, the protein is purified successfully, then antibody is to be raised against it for
finding its localization by immunoflurescence microscopy. Enzyme assay, also to be done to
know its activity .

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