This document summarizes work done on culturing crocodile cell lines and cloning the parc gene from Pseudomonas keratitis. Primary crocodile cell lines were established from various organs and immortalized using hTERT. The parc gene was cloned from mutant and wild-type Pseudomonas strains and will be expressed and crystallized to study its role in quinolone antibiotic resistance.

1. WORK REPORT
A) CULTURING OF CROCODILE CELL LINES.
B) CLONING AND EXPRESSION OF parc GENE (Pseudomonas keratitis)

2. A) TISSUE CULTURE
1) Establishing primary cell lines of crocodile.
2) Immortalization of crocodile cell lines.

3. 1) Establishing primary cell line of crocodile
Dissected organs were chopped and cells were allowed to adhere to the surface of culture dish.
Cell growth was optimized by different media i.e. DMEM, RPMI, IMDM and incubator
temperature maintained at 28±0.5
Cells were subsequently passaged when confluent and back up culture freezed in liquid nitrogen.
i.e. Brain, Heart, Liver, Kidney, Genital ridge
Currently: Brain cells being maintained at P11 and P12 in DMEM(15%) .
Total no. of freezed cells : 158

4. Male21 genital ridge P0
Female 22 brain P0 Male 21 genital ridge P0
Male 21 heart P0

5. Male21 kidney P0 Male 21 kidney P0
Male21 liver P0

6. Brain Female 25 P9

7. Heart male 27 P8

8. Liver Female 25 P4

9. a) Immortalization using chemical method i.e. Niso4. (unsuccessful)
b) Immortalization using molecular approach i.e. hTERT. (constructs being prepared)
2) Immortalization of the crocodile cell line
pEGFP-N1 Vector having hTERT fragment (Ready) but to be modified.
pTRIP-Vector having hTERT fragment ( under construction)
pDSRed2-C1 having hTERT fragment (by Nawaz)
pEGFP-N1 Vector carrying CAG- promoter
c) To do Transfection studies in cells using the above vectors.
d) To support the data consistency in transfection ,establish karyotyping and possible
pcr assays.

10. VECTOR
INSERT

11. a) Plasmid of pEGFP –N1 4.7 Kb b) Restriction digestion of vector
EcoRI SalI EcoRI uncut
+SalI
1Kb
d) Ligation check
e) Restriction digestion
of vector with insert
f) Touchdown pcr with
hTERT primers
1Kb
1Kb
1Kb
c) Restriction digestion
of hTERT (insert) 3.5kb
pEGFP-N1 with hTERT
Eluted
and given
for sequencing
EcoRI
+SalI
Expected
8.2 Kb
EcoRI+ SalI

12. c) Restriction digestion
of hTERT (insert) 3.5kb
a) Plasmid of lentiviral vector 9.6Kb
1Kb
b) Restriction digestion of vector
1KbSmaI Uncut
1Kb
Conversion of sticky ends
of insert into blunt ends
by Klenow Polymerase
EcoRI
+SalI
pTRIP-Vector with hTERT
1Kb
d) Ligation check
Expected
13 kb
6:1 8:1
1Kb6:1 8:1
e) Restriction digestion of
transformed plasmid

13. pEGFP-N1 Vector carrying CAG- promoter
Plasmid Transformation is done , yet to start with cloning procedures

14. TRANSFECTION EXPERIMENT WITH pEGFP-N1
Plated 6 well plate ( having cells from 1 confluent T-25 flasks)
i.e f_25+Brain P₁₁
Next day added construct and lipofectamine in 2:1 to 3 wells ,2 wells
maintained as blank and one with only GFP.
Allowed the plate to be in serum free media for 4 hrs
After completion of 4 hrs added serum containing medium to the 3 wells
2 days later checked under fluorescence microscope
RESULT: High cell mortality , cells seen as transparent and floating

15. C) Expression of parc gene from ( Pseudomonas keratitis)
Pseudomonas keratitis is a human pathogen and is intrinsically resistant to a wide variety of
antibiotics.
The major mechanisms of bacterial resistance to quinolones are the modifications of the target
site of DNA gyrase and Topoisomerase IV.
Although many studies have focused on DNA gyrase, studies on Topoisomerase IV are less
advanced for quinolone resistant Pseudomonas species.
To study the importance of hydrophilic amino acid change to hydrophobic amino acid TCG –
TGG (Serine to Tryptophan) which we suppose may help in acquiring resistance to quinolone
group of antibiotics.
To study the above role we aim to express the parc gene from the mutant and crystallize it to
study the interaction of amino acids as how they impart the resistance.

16. Total 8 strains of sample were given to screen for the mutants .
DNA isolation was done and pcr with partial parc primers that gave 186 bp region.
Work Done
After screening chose randomly 1312 (mutant) and 1353 (wild) for cloning purpose.
Sequencing for the samples revealed the following data:
a)1353,2047,2162,1323 (WILD)
b)1312,2051,2045,1378 (MUTANTS)

17. GGATCCATGAGCGAATCCCTCGATCTGAGCCTGGAAGGGGTCGAACGCCGGTCGTTGGCCGAGTTCACCGAGC
AGGCCTATCTGAACTATTCCATGTACGTGATCATGGACCGCGCCCTGCCGCATATCGGCGACGGCCTGAAACCGGTGCA
GCGACGCATCGTCTACGCCATGAGCGAACTGGGGCTGGATGCCGATTCCAAGCACAAGAAGTCGGCGCGCACCGTCG
GCGACGTGCTCGGCAAGTTCCACCCGCACGGCGACTCGGCCTGCTACGAGGCCATGGTGCTGATGGCGCAGCCGTTCT
CCTATCGCTATCCGCTGGTGGACGGCCAGGGCAACTGGGGGGCTCCGGACGATCCCAAGTCCTTCGCCGCCATGCGTT
ATACCGAGGCGCGCCTGTCGCGCTATTCCGAGGTGCTGCTCAGCGAACTGGGCCAGGGTACCGTGGACTGGGTACCGA
ACTTCGACGGCACCCTCGACGAGCCGGCCGTGCTGCCGGCCCGCCTGCCCAACCTGCTGCTCAACGGCACCACCGGCAT
CGCGGTGGGCATGGCCACCGACGTGCCGCCGCACAACCTGCGGGAAGTCGCGTCGGCCTGCGTGCGCCTGCTCGACC
AGCCGGGCGCGACGGTCGCCGAATTGTGCGAACACGTGCCGGGCCCGGACTTCCCCACCGAAGCCGAGATCATCACCC
CGCGCGCCGACCTGCAGAAGGTCTACGAGACCGGCCGCGGTTCGGTGCGCATGCGCGCGGTGTACCGCTTCGAGGAC
GGCGATATCGTCATCCACGCCCTGCCGCACCAGGTGTCCGGTTCCAAGGTGCTGGAACAGATCGCCGGGCAGATGCAG
GCCAAGAAGCTGCCGATGGTGGCCGACCTGCGCGACGAGTCGGACCACGAGAACCCGACCCGCATCGTCATCATCCCG
CGTTCGAACCGGGTCGATGTCGAAGAGCTGATGACCCATCTGTTCGCCACCACCGACCTGGAGACCAGCTACCGGGTC
AACCTGAACATCATCGGCCTCGACGGCAAGCCGGCAGTCAAGGACCTGCGCCAGTTGCTCTCGGAGTGGCTGCAGTTC
CGCATCGGCACCGTGCGTCGACGCCTGCAGTTCCGCCTGGACAAGGTCGAGCGCCGCCTGCATCTGCTGGATGGCTTG
CTGATCGCCTTCCTCAACCTCGACGAGGTGATCCACATCATCCGCACCGAGGACCAGCCCAAGGCGGTGCTGATGGAG
CGCTTCGAACTCAGCGAGGTGCAGGCCGACTACATCCTCGACACCCGCCTGCGCCAGTTGGCACGCCTGGAAGAGATG
AAGATCCGCGGCGAGCAGGAAGAGTTGCTGAAGGAGCAGAAGCGCCTGCAGACCCTGCTCGGCAGCGAGGCCAAGC
TGAAGAAGCTGGTGCGCGAGGAGCTGATCAAGGACGCCGAGACCTACGGCGACGACCGCCGTTCGCCGATCGTCGCC
CGCGCCGAGGCCCGCGCGCTGTCGGAAACCGAGCTGATGCCCACCGAACCGGTGACCGTGGTGCTCTCGGAAAAAGG
CTGGGTGCGTTGCGCCAAGGGCCACGACATCGACGCCGCCGGCCTCTCCTACAAGGCCGGCGACGGCTTCAAGGCCGC
CGCGCCGGGACGCTCGAACCAGTATGCGGTGTTCATCGACTCCACCGGGCGCAGCTACTCGCTGCCGGCCCACAGCCT
GCCGTCCGCGCGAGGCCAGGGCGAGCCACTCAGCGGCCGGCTGACGCCGCCGCCGGGGGCCAGCTTCGAATGCGTGC
TGCTGCCGGACGACGATGCGCTGTTCGTGATCGCTTCCGACGCCGGCTATGGTTTCGTGGTCAAGGGCGAGGACCTGC
AGGCCAAGAACAAGGCCGGCAAGGCCCTGCTCAGCCTGCCCAACGGCTCCGCCGTGGTGGCGCCGCGCCCGGTGCGC
GATGTGGAGCAGGATTGGCTGGCGGCCGTGACGACCGAGGGCCGTCTGCTATTGTTCAAGGTCTCCGACCTGCCGCAG
CTCGGCAAGGGCAAGGGCAACAAGATCATCGGCATCCCCGGCGAACGCGTGGCCAGCCGCGAGGAATACCTCACCGA
CCTGGCTGTTCTGCCAGCCGGGGCGACGTTGGTCCTGCAGGCCGGAAAGCGTACCCTGTCGCTCAAGGGCGACGACCT
GGAACACTACAAGGGGGAGCGAGGCCGGCGAGGCAACAAGCTGCCGCGCGGTTTCCAGCGCGTCGACAGCCTGCTG
GTGGATATTCCGCCACAGGATTGACTCGAGCACCACCACCACCACCACTGAGATCCGGCT
Forward primer
5’-tttGGATCCATGAGCGAATCCCTCGATCTG-3’
Reverse primer
5’-ttatCTCGAGTCAATCCTGTGGCGGAATATC-3’
Complete size of gene
2.3 Kb
Mol wt. of Protein
186 kDa
BamHI
XhoI

19. a) DNA isolation
L 1312 1353
b)Pcr with full length primers
2.5 kb
1kb 1312 1353
c) Digestion of inserts
1312 1312 1353 1353 1kb
Bam+Xho Uncut uncut Bam+Xho
d)Plasmid of Pet28
5.4 kb
e) Digestion of vector
Bam Xho Bam+Xho 1kb uncut
f) Ligation pcr
1312 1353 1kb
1kb Expected 2.3 kb

20. 1kb
1kb
1312 samples 1353 samples
COLONY PCR RESULTS
1kb