Experiment 2 Report

Clone the V.harveyi chb gene from an

SDS isolate into pUC19 to make a new
recombinant plasmid (pBMB2)








Soojung Kim
4/15/14

BMB445W Section 2
Soojung Kim


References
1. Ingalls, A. M., M. M. Dickie, and G. D. Snell. 1950. Obese, a new mutation in the house mouse. The Journal of
heredity 41: 317-318.
2. Zhang, Y., R. Proenca, M. Maffei, M. Barone, L. Leopold, and J. M. Friedman. 1994. Positional cloning of the
mouse obese gene and its human homologue. Nature 372: 425-432

Experiment 2 Part A Write Up

The OB Gene
The obese (OB) gene and its product leptin are involved in the regulation of both appetite and
energy expenditure in the mouse. In 1950, the OB gene was discovered in the Jackson
Laboratory when the ob/ob mouse arose by chance in a colony. The ob/ob genotype mice (two
defective copies of the ob gene; lowercase letters signify a mutant for of the gene) show the
behavior and physiology of animals in a constant state of starvation: they are unable to stay
warm and they grow abnormally, exhibit uncontrolled appetite, and do not reproduce. As a result
of their unrestrained consumption of food, these mutant mice become severely obese, weighing
as much as three times more than normal mice
(Figure 1; Ingalls et al., 1950).

Figure 1: Obesity caused in ob/ob genotype mice: Both of these
mice, which are the same age show difference in body mass and
weight, a result of the mutation in the OB gene.





Leptin is a product of the OB gene that was discovered in 1994 by the Friedman laboratory.
(Zhang et al.,1994) As an adipokine protein hormone, leptin (167 amino acids) acts on receptors
in the hypothalamus, as it reaches the brain, to curtail appetite. Leptin, together with the leptin
receptor work together to regulate feeding behavior ; leptin carries the message that fat reserves
are sufficient, and it promotes a reduction in fuel intake and increased expenditure of energy.
Leptin-receptor interaction in the hypothalamus changes the release of signals to the brain that
influences appetite. Leptin also stimulates the sympathetic nervous system, increasing blood
pressure, heart rate, and thermogenesis to permit oxidation of fuel, dissipating energy as heat and
consuming calories. In Friedmans experiment, two strains of mice both having defects in OB
gene were compared. It was found that the mouse strain injected regularly with purified leptin
maintained normal weight, while the mouse strain that received no leptin failed to limit its
appetite and became grotesquely fat. This confirmed leptins role in the physiological system that
regulates food intake and metabolism. The Friedman lab was able to clone the OB gene in mice
and its homolog in humans. OB is known to encode a 4.5-kilobase (kb) adipose tissue messenger
RNA with ahighly conserved 167-amino-acid open reading frame. The predicted amino-acid is
84% identical between human and mouse. The purpose of cloning the OB gene is to further
study the robust physiologic system that regulates food intake and metabolism. Our laboratory
team wants to analyze the OB gene and its product Leptin in order to develop a dietary
supplement that will enable regulation of the Leptin levels so that obesity can be treated like any
other disorder in a health-care setting. Similar to the cancer drugs developed, obesity must be
recognized as a problem of biology and effective drugs and treatment methods must be
developed. Further study of the OB gene and Leptin will allow such research and advancement.


Determining the details of cloning the OB (LEP) Gene
The OB (LEP) gene nucleotide sequence can be found from the NCBI Entrez search engine (GI:
3952). This whole genome is located on Chromosome 7 and spans from nucleotide 128241189 to
128257629. This whole gene, which spans a length of 16440 base-pairs, is too large to clone into
a cloning vector. Thus, a portion of the gene, the Homo sapiens leptin (LEP), mRNA (Acession #:
NM_000230.2) will be used for the cloning of the OB gene. The leptin mRNA is 3444 base-pair-
long and includes the coding sequence of the leptin protein; a highly conserved 167- amino acid
long region (58 561 bp). Because this particular sequence is derived from a human gene, we
want the cloning to utilize the expression in mammalian cells, and thus will be using the
pcDNA3.1 (+) vector.


Figure 2: pcDNA3.1 Vector The restriction enzymes sites
within the MCS of the vector are shown with the vector map.
















This cloning method uses restriction enzyme and DNA ligase approach to clone the OB gene
into the expression plasmid pcDNA3.1(+). Two restriction enzyme sites are used at each end of
the LEP mRNA sequence to allow for directional cloning into pcDNA3.1(+). In PCR-based
cloning, it is important to select restriction enzymes that will cleave within the multiple cloning
site of the vector, in this case the pcDNA 3.1 (+), but not within the original target gene to be
cloned. The NEBcutter tool is used to determine the 0 cutters for the LEP mRNA sequence,
which gives a list of 71 possible noncutters for the sequence. These 71 restriction enzymes are
compared to the restriction sites within pcDNA3.1(+) to determine the optimal restriction
enzymes used for the double digest. Of the possible restriction enzymes, NheI and EcoRV are
selected because the two are farthest away from each other within the MCS. Both NheI and
EcoRV are heat inactivatable and their cleavage activites are not affected by methylation status.
Also, both enzymes of the high fidelity version, and Double Digest Finder reveals that a double
digest reaction using Nhe1-HF and EcoRV-HF is optimal in CutSmart
TM
Buffer at 37C, with
both enzymes exhibiting 100% activity in this setup.
The primers used for this cloning procedure are designed following the Best Practice Guidelines
for Primer Design. We want the pcDNA3.1(+) promoter to drive the gene expression instead of

the endogenous promoter. Therefore, the NheI restriction enzyme sequences are included as the
forward primer and EcoRV restriction sequences as the reverse primer so that the LEP mRNA
sequence is positioned in-frame with the promoter. Six to seven random nucleotides are added to
each end of the restriction sequences for each primer. For the reverse primer, using the LEP
mRNA 3 end sequences was impossible because the last 17 base-pairs of the LEP mRNA were
a repetition of Adenine sequences. Thus, the reverse primer was designed to bind to nucleotide
3352 3361, instead of the very end sequences of mRNA at nucleotide 3444 bp. The melting
temperatures, GC content, and secondary structure of primers were checked with OligoCalc
program. The designed primers are listed below:
Forward Primer: 5-GCACATAGCTAGCGTAGGAATCGC-3
Reverse Primer: 5-CGGAATCTCGGATATCGAATGC-3

The Cloning Procedure
1) The LEP mRNA sequence from the whole OB gene will be amplified out from the
forward and reverse primers designed. PCR mixture includes: the template OB gene
DNA, Forward and Reverse Primers, PCR Master Mix (2X), dNTPs.
The Temperature incubations programmed so that the first few cycles have a
lower annealing temperature around 50C. This allows the primers, which are not
completely complimentary to the OB gene, to correctly anneal to initiate the first
few cycles of copying. After 5-6 cycles, the annealing temperature is raised to
around 57C to speed up the amplification process.
o Sample Program:
94 C
50C
72C
4 min
1 min
7 min
94 C
50C
72C
1 min |
1 min | (Repeat for 5 cycles)
7 min |
94 C
58C
72C
1 min |
1 min | (Repeat for 20 cycles)
7 min |
94 C
58C
72C
4 min
1 min
7 min
4C 24 hours
The polymerase of choice in this PCR reaction is the Phusion Polymerase,
because we want the amplification of the gene to go through as accurately as
possible. Phusion has a DNA binding domain that increases processivity, which is
advantageous in amplifying longer targets such as this LEP mRNA sequence. The
Phusion Polymerase also has proofreading ability to ensure accuracy. The PCR
program at 72C runs 7 minutes because Phusion takes longer per kilobase to
amplify.
2) The PCR product will be purified away from the PCR buffer, dNTPs, polymerase and
primers using solution purification method. Gel-electrophoresis will be used to analyze
the purified product.

3) Both the purified PCR product and the pcDNA3.1(+) vector will be double digested
separately with PmeI and CutSmart
TM
Buffer at 37C.
4) The double digested pcDNA3.1(+) vector will be phosphatase treated to avoid self
relegation. Double digested PCR and vector will be heat inactivated and analyzed using
gel-electrophoresis.
5) Ligation reactions will be set up:
Control: includes T4 DNA Ligase Master Mix and Vector DNA but does not
include the Insert DNA
Experimental: Insert DNA and Vector DNA are mixed in the presence of T4
DNA Ligase Master Mix. The relative volumes of the vector and insert are
determined using the following equation
o Li/Lv = 3*(Si/Sv) *(Iv/Ii) *(Vi/Vv)
6) After Ligation reaction is completed, the Kill Cut procedure will be utilized. The PmeI
restriction enzyme cuts the MCS of the pcDNA3.1(+) vector but does not affect the LEP
mRNA sequence. Thus, PmeI is used as a Kill Cut RE of the ligation product.
7) Transformation reactions are set up using the ligation products.
Ligation reactions are transformed into a mammalian cell to allow amplification
and isolation
Ligation control reactions are also transformed into mammalian cell
Transformation controls are set up with no DNA for negative control and
undigested pcDNA3.1(+) for the positive control
The selection will be enabled by the use of antibiotic resistance.
PCR cloning using primers complementary to the OB gene LEP mRNA insert
will be used to screen for true positive colonies.






















Appendix

3/20/14 Homo sapiens leptin (LEP), mRNA - Nucleotide - NCBI
www.ncbi.nlm.nih.gov/nuccore/NM_000230.2 1/4
Homo sapiens Ieptin (LEP), mRNA
NCB Reference Sequence: NM_000230.2
FASTA Graphics
Go to:
LOCUS NM_000230 3444 bp mRNA linear PRI l6~MAR~20l4
DEFINITION Homo sapiens leptin {LEP, mRNA.
ACCESSION NM_000230
VERSION NM_000230.2 GI:l69790920
KEYWORDS RefSeq.
SOURCE Homo sapiens {human
ORGANISM Homo sapiens
Eukaryota, Metazoa, Chordata, Craniata, Vertebrata, Euteleostomi,
Mammalia, Eutheria, Euarchontoglires, Primates, Haplorrhini,
Catarrhini, Hominidae, Homo.
REFERENCE l {bases l to 3444
AUTHORS Hara K, Fuita H, Johnson TA, Yamauchi T, Yasuda K, Horikoshi M,
Peng C, Hu C, Ma RC, Imamura M, Iwata M, Tsunoda T, Morizono T,
Shoima N, So WY, Leung TF, Kwan P, Zhang R, Wang J, Yu W, Maegawa
H, Hirose H, Kaku K, Ito C, Watada H, Tanaka Y, Tobe K, Kashiwagi
A, Kawamori R, Jia W, Chan JC, Teo YY, Shyong TE, Kamatani N, Kubo
M, Maeda S and Kadowaki T.
CONSRTM DIAGRAM consortium
TITLE Genome~wide association study identifies three novel loci for type
2 diabetes
JOURNAL Hum. Mol. Genet. 23 {l, 239~246 {20l4
PUBMED 23945395
REFERENCE 2 {bases l to 3444
AUTHORS Cundrle,I. Jr., Somers,V.K., Singh,P., Johnson,B.D., Scott,C.G.,
van der Walt,C. and Olson,L.J.
TITLE Leptin deficiency promotes central sleep apnea in patients with
heart failure
JOURNAL Chest l45 {l, 72~78 {20l4
PUBMED 24030529
REMARK GeneRIF: In patients with heart failure {HF and central sleep
apnea, leptin concentration is low and is inversely related to
natriuretic peptide {NP concentration. Counterregulatory
interactions of leptin and NP may be important in ventilatory
control in HF.
REFERENCE 3 {bases l to 3444
AUTHORS Hart CN, Carskadon MA, Considine RV, Fava JL, Lawton J, Raynor HA,
Jelalian E, Owens J and Wing R.
TITLE Changes in children`s sleep duration on food intake, weight, and
leptin
JOURNAL Pediatrics l32 {6, El473~El480 {20l3
PUBMED 24l90680
REMARK GeneRIF: Compared with decreased sleep, increased sleep duration in
school~age children resulted in lower reported food intake, lower
fasting leptin levels, and lower weight.
REFERENCE 4 {bases l to 3444
AUTHORS Kleinridders,A., Lauritzen,H.P., Ussar,S., Christensen,J.H.,
Mori,M.A., Bross,P. and Kahn,C.R.
TITLE Leptin regulation of Hsp60 impacts hypothalamic insulin signaling
JOURNAL J. Clin. Invest. l23 {ll, 4667~4680 {20l3
PUBMED 24084737
REMARK GeneRIF: In type 2 diabetes, there is hypothalamic insulin
resistance and mitochondrial dysfunction due to downregulation of
the mitochondrial chaperone HSP60. HSP60 reduction was due to a
lack of proper leptin signaling.
REFERENCE 5 {bases l to 3444
AUTHORS Pathak RR, Grover A, Malaney P, Quarni W, Pandit A, Allen~Gipson D
and Dave V.
TITLE Loss of phosphatase and tensin homolog {PTEN induces
leptin~mediated leptin gene expression: feed~forward loop operating
in the lung
JOURNAL J. Biol. Chem. 288 {4l, 2982l~29835 {20l3
PUBMED 23963458
REMARK GeneRIF: Data indicate that loss of Pten in the lung epithelial
cells in vivo activated adipokine signaling and induced leptin
synthesis.
REFERENCE 6 {bases l to 3444
DispIay Settings: GenBank
NucIeotIde
CDS Feature 1 of 1 NM_000230 : 1 segment
Display: FASTA GenBank Help
Details

Sequence of the portion of LEP gene that will be cloned:
GTAGGAATCGCAGCGCCAGCGGTTGCAAGGCCCAAGAAGCCCATCCTGGGAAGGAAAATGCATTGGGGAA
CCCTGTGCGGATTCTTGTGGCTTTGGCCCTATCTTTTCTATGTCCAAGCTGTGCCCATCCAAAAAGTCCA
AGATGACACCAAAACCCTCATCAAGACAATTGTCACCAGGATCAATGACATTTCACACACGCAGTCAGTC
TCCTCCAAACAGAAAGTCACCGGTTTGGACTTCATTCCTGGGCTCCACCCCATCCTGACCTTATCCAAGA
TGGACCAGACACTGGCAGTCTACCAACAGATCCTCACCAGTATGCCTTCCAGAAACGTGATCCAAATATC
CAACGACCTGGAGAACCTCCGGGATCTTCTTCACGTGCTGGCCTTCTCTAAGAGCTGCCACTTGCCCTGG
GCCAGTGGCCTGGAGACCTTGGACAGCCTGGGGGGTGTCCTGGAAGCTTCAGGCTACTCCACAGAGGTGG
TGGCCCTGAGCAGGCTGCAGGGGTCTCTGCAGGACATGCTGTGGCAGCTGGACCTCAGCCCTGGGTGCTG
AGGCCTTGAAGGTCACTCTTCCTGCAAGGACTACGTTAAGGGAAGGAACTCTGGCTTCCAGGTATCTCCA
GGATTGAAGAGCATTGCATGGACACCCCTTATCCAGGACTCTGTCAATTTCCCTGACTCCTCTAAGCCAC
TCTTCCAAAGGCATAAGACCCTAAGCCTCCTTTTGCTTGAAACCAAAGATATATACACAGGATCCTATTC
TCACCAGGAAGGGGGTCCACCCAGCAAAGAGTGGGCTGCATCTGGGATTCCCACCAAGGTCTTCAGCCAT
CAACAAGAGTTGTCTTGTCCCCTCTTGACCCATCTCCCCCTCACTGAATGCCTCAATGTGACCAGGGGTG
ATTTCAGAGAGGGCAGAGGGGTAGGCAGAGCCTTTGGATGACCAGAACAAGGTTCCCTCTGAGAATTCCA
AGGAGTTCCATGAAGACCACATCCACACACGCAGGAACTCCCAGCAACACAAGCTGGAAGCACATGTTTA
TTTATTCTGCATTTTATTCTGGATGGATTTGAAGCAAAGCACCAGCTTCTCCAGGCTCTTTGGGGTCAGC
CAGGGCCAGGGGTCTCCCTGGAGTGCAGTTTCCAATCCCATAGATGGGTCTGGCTGAGCTGAACCCATTT
TGAGTGACTCGAGGGTTGGGTTCATCTGAGCAAGAGCTGGCAAAGGTGGCTCTCCAGTTAGTTCTCTCGT
AACTGGTTTCATTTCTACTGTGACTGATGTTACATCACAGTGTTTGCAATGGTGTTGCCCTGAGTGGATC
TCCAAGGACCAGGTTATTTTAAAAAGATTTGTTTTGTCAAGTGTCATATGTAGGTGTCTGCACCCAGGGG
TGGGGAATGTTTGGGCAGAAGGGAGAAGGATCTAGAATGTGTTTTCTGAATAACATTTGTGTGGTGGGTT
CTTTGGAAGGAGTGAGATCATTTTCTTATCTTCTGCAATTGCTTAGGATGTTTTTCATGAAAATAGCTCT
TTCAGGGGGGTTGTGAGGCCTGGCCAGGCACCCCCTGGAGAGAAGTTTCTGGCCCTGGCTGACCCCAAAG
AGCCTGGAGAAGCTGATGCTTTGCTTCAAATCCATCCAGAATAAAACGCAAAGGGCTGAAAGCCATTTGT
TGGGGCAGTGGTAAGCTCTGGCTTTCTCCGACTGCTAGGGAGTGGTCTTTCCTATCATGGAGTGACGGTC
CCACACTGGTGACTGCGATCTTCAGAGCAGGGGTCCTTGGTGTGACCCTCTGAATGGTCCAGGGTTGATC
ACACTCTGGGTTTATTACATGGCAGTGTTCCTATTTGGGGCTTGCATGCCAAATTGTAGTTCTTGTCTGA
TTGGCTCACCCAAGCAAGGCCAAAATTACCAAAAATCTTGGGGGGTTTTTACTCCAGTGGTGAAGAAAAC
TCCTTTAGCAGGTGGTCCTGAGACCTGACAAGCACTGCTAGGCGAGTGCCAGGACTCCCCAGGCCAGGCC
ACCAGGATGGCCCTTCCCACTGGAGGTCACATTCAGGAAGATGAAAGAGGAGGTTTGGGGTCTGCCACCA
TCCTGCTGCTGTGTTTTTGCTATCACACAGTGGGTGGTGGATCTGTCCAAGGAAACTTGAATCAAAGCAG
TTAACTTTAAGACTGAGCACCTGCTTCATGCTCAGCCCTGACTGGTGCTATAGGCTGGAGAAGCTCACCC
AATAAACATTAAGATTGAGGCCTGCCCTCAGGGATCTTGCATTCCCAGTGGTCAAACCGCACTCACCCAT
GTGCCAAGGTGGGGTATTTACCACAGCAGCTGAACAGCCAAATGCATGGTGCAGTTGACAGCAGGTGGGA
AATGGTATGAGCTGAGGGGGGCCGTGCCCAGGGGCCCACAGGGAACCCTGCTTGCACTTTGTAACATGTT
TACTTTTCAGGGCATCTTAGCTTCTATTATAGCCACATCCCTTTGAAACAAGATAACTGAGAATTTAAAA
ATAAGAAAATACATAAGACCATAACAGCCAACAGGTGGCAGGACCAGGACTATAGCCCAGGTCCTCTGAT
ACCCAGAGCATTACGTGAGCCAGGTAATGAGGGACTGGAACCAGGGAGACCGAGCGCTTTCTGGAAAAGA
GGAGTTTCGAGGTAGAGTTTGAAGGAGGTGAGGGATGTGAATTGCCTGCAGAGAGAAGCCTGTTTTGTTG
GAAGGTTTGGTGTGTGGAGATGCAGAGGTAAAAGTGTGAGCAGTGAGTTACAGCGAGAGGCAGAGAAAGA
AGAGACAGGAGGGCAAGGGCCATGCTGAAGGGACCTTGAAGGGTAAAGAAGTTTGATATTAAAGGAGTTA
AGAGTAGCAAGTTCTAGAGAAGAGGCTGGTGCTGTGGCCAGGGTGAGAGCTGCTCTGGAAAATGTGACCC
AGATCCTCACAACCACCTAATCAGGCTGAGGTGTCTTAAGCCTTTTGCTCACAAAACCTGGCACAATGGC
TAATTCCCAGAGTGTGAAACTTCCTAAGTATAAATGGTTGTCTGTTTTTGTAACTTAAAAAAAAAAAAAA
AAGTTTGGCCGGGTGCGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCAAGGTGGGGGGATCAC
AAGGTCACTAGATGGCGAGCATCCTGGCCAACATGGTGAAACCCCGTCTCTACTAAAAACACAAAAGTTA
GCTGAGCGTGGTGGCGGGCGCCTGTAGTCCCAGCCACTCGGGAGGCTGAGACAGGAGAATCGCTTAAACC
TGGGAGGCGGAGAGTACAGTGAGCCAAGATCGCGCCACTGCACTCCGGCCTGATGACAGAGCGAGATTCC
GTCTTAAAAAAAAAAAAAAAAAAGTTTGTTTTTAAAAAAATCTAAATAAAATAACTTTGCCCCCTGCAAA
AAAAAAAAAAAAAA


This is the sequence of the LEP mRNA sequence, which is 3444 bp-long in total. The 504 bp
region from 58 to 561 bp is the CDS of the gene that corresponds to the Leptin product. The
CDS of the mRNA is marked in figure below as reference.




Primer Design

Forward: 5 GCACATA GCTAGC GTAGGAATCGC 3
Extra Bases NheI-HF

Reverse: 5 CGTAAG GATATC CGGAATCTCG 3
Extra Bases EcoRV-HF


5/1/14 NEBcutter
tools.neb.com/NEBcutter2/listbycuts.php?name=47363406-&numcuts=0 1/2
Enzymes that don't cut

unnamed sequence
Help Comments
[Back to main
display]
Number of cuts = 0 OK Save as text IIIe
# Enzyme Specificity
1 Aat G ACGT C
2 Acc65 G GTAC C
3 Acl AA CG TT
4 Ahd GACNN N NNGTC
5 ApaL G TGCA C
6 Asc GG CGCG CC
7 Ase AT TA AT
8 AsiS GCG AT CGC
9 Avr C CTAG G
10 Bae
{N
5
{N
l0
ACNNNNGTAYC{N
7
{N
5
11 Bcg
NN {N
l0
CGA{N
6
TGC{N
l0
NN
12 BciV
GTATCC{N
5
N
13 Bgl GCCN NNN NGGC
14 Bgl A GATC T
15 BmgB CAC GTC
16 Bmt G CTAG C
17 BpuE
CTTGAG{N
l4
NN
18 BsaB GATNN NNATC
19 BsiE CG RY CG
20 BsiW C GTAC G
21 BspD AT CG AT
22 BspE T CCGG A
23 BsrB CCG CTC
24 BsrG T GTAC A
25 BssH G CGCG C
26 BssS C ACGA G
27 BstB TT CG AA
28 BstE G GTNAC C
29 BstZ17 GTA TAC
30 Btg C CRYG G
31 BtgZ
GCGATG{N
l0
NNNN
32 Cla AT CG AT
33 CspC
NN {N
ll
CAA{N
5
GTGG{N
l0
NN
34 Drd GACNN NN NNGTC
5/1/14 NEBcutter
tools.neb.com/NEBcutter2/listbycuts.php?name=47363406-&numcuts=0 2/2
35 Eag C GGCC G
36 Eco53k GAG CTC
37 EcoRV GAT ATC
38 Fse GG CCGG CC
39 Fsp TGC GCA
40 Hga
GACGC{N
5
{N
5
41 Hpy99 CGWCG
42 Kpn G GTAC C
43 Mlu A CGCG T
44 Nae GCC GGC
45 Nco C CATG G
46 NgoMV G CCGG C
47 Nhe G CTAG C
48 NmeA
GCCGAG{N
l9
NN
49 Not GC GGCC GC
50 Nru TCG CGA
51 Pac TTA AT TAA
52 Pme GTTT AAAC
53 Psi TTA TAA
54 Pvu CG AT CG
55 Rsr CG GWC CG
56 Sac G AGCT C
57 Sac CC GC GG
58 Sal G TCGA C
59 Sbf CC TGCA GG
60 Sca AGT ACT
61 Sfi GGCCN NNN NGGCC
62 SgrA CR CCGG YG
63 Sma CCC GGG
64 SnaB TAC GTA
65 Spe A CTAG T
66 Ssp AAT ATT
67 Swa ATTT AAAT
68 TspM C CCGG G
69 Xma C CCGG G
70 Xmn GAANN NNTTC
71 Zra GAC GTC
|omo lroducts lhol-||t

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Product udato: A troo viaI ot CoI Loading Dyo, PurIo (0x) ia now incIudod with aII EF roatriction onzymoa. PIoaao noto thoro may
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stra|ghttorward and stroam||nod samp|o procoss|ng. || onzymos a|so oxh|b|t dramat|ca||y roducod star act|v|ty. || onzymos aro a|| 1|mo-
3avor qua||t|od and can thorotoro cut substrato 0lA |n b-1b w|th tho t|ox|b|||ty to d|gost ovorn|ght w|thout dogradat|on to 0lA. Eng|noorod
w|th portormanco |n m|nd, || rostr|ct|on onzymos aro tu||y act|vo undor a broador rango ot cond|t|ons, m|n|m|z|ng ott-targot products, wh||o
ottor|ng t|ox|b|||ty |n oxpor|monta| dos|gn.
Product 5ourco
An stra|n that carr|os tho c|onod and mod|t|od (010A, E27A) Ecolv gono trom tho p|asm|d 102 plG74 (l.l. G|atman)
Roagonta 5uIiod
1ho to||ow|ng roagonts aro supp||od w|th th|s product:
|omo 1oo|s 8 losourcos lntoract|vo 1oo|s 0oub|o 0|gost ||ndor
Dou|ie Digest liucer
Wo havo oxciting nowaI Wo havo tranaitionod our roatriction onzymoa into a now buttor ayatom in ordor to mako your roatriction
digoata ovon moro convoniont tor you. 1h|s |atost |mprovomont |nvo|vos tho add|t|on ot 33A, as wo|| as tho romova| ot 011. 3y add|ng
33A to tho buttor, wo aro now ab|o to ottor ~200 onzymos that cut |n a s|ng|o buttor. 1h|s |mprovos oaso-ot-uso, ospoc|a||y whon portorm|ng
doub|o d|gosts. lt a|so o||m|natos tho nood to add 33A soparato|y. lE3uttors 1, 2 and 8 havo boon rop|acod by lE3uttors 1.1, 2.1 and 8.1,
rospoct|vo|y. lE3uttor 4 has boon rop|acod w|th Cut3mart 3uttor. l|oaso noto that tho o|d buttors aro st||| ava||ab|o. 0ota||od |ntormat|on on
tho now buttor systom, a|ong w|th an oxtons|vo ||st ot |AOs, can bo tound at lE3Cut3mart.com.
1ho 0oub|o 0|gost ||ndor now conta|ns |ntormat|on on tho now buttor systom. |owovor, wo undorstand that many customors w||| st||| havo
onzymos that woro prov|dod w|th tho o|d buttor systom and w||| thus st||| nood to rotoronco th|s |ntormat|on. 1ho o|d' 0oub|o 0|gost ||ndor |s
ava||ab|o on oach rostr|ct|on onzymo product pago. You can got add|t|ona| support by contact|ng |ntoQnob.com.
Uso th|s too| to gu|do your roact|on buttor so|oct|on whon sott|ng up doub|o d|gosts, a common t|mosav|ng procoduro. Choos|ng tho r|ght
buttors w||| ho|p you to avo|d star act|v|ty and |oss ot product.
loarn moro |ntormat|on about doub|o d|gosts, |nc|ud|ng how to sot up a roact|on.
1ho o|d Act|v|ty/lortormanco Chart |s ava||ab|o on oach rostr|ct|on onzymo product pago and trom tho now lE3uttor Act|v|ty/lortormanco
Chart.
3o|oct 1st onzymo
lhol-||t
3o|oct 2nd onzymo
Ecolv-||t
Co Co
Enzymo Cat # 1om 5uIiod NEButtor 5uIomonta Activity in NEButtor
5AN 1.1 2.1 8.1 Cut5mart
lhol-||t l8181 87C Cut3mart" 3uttor no 100 2b 10 100
Ecolv-||t l810b 87C Cut3mart" 3uttor no 2b 100 100 100
Dou|ie Digest keeonneucatious ior rbeI-ul+ LeokV-ul+.
0|gost |n Cut3mart" 3uttor at 87C.
loarn moro about |mprovomonts to tho lE3 lostr|ct|on Enzymo 3uttor 3ystom

Forward Primer: 5-GCACAGCGCTAGCGTAGGAATCGC-3

Primer Properties from Oligocalc:





Reverse Primer: 5-CGTAAGGATATCCGGAATCTCG -3

Primer Properties from Oligocalc:



Experiment 2 Part B

Introduction
In order to understand a complex biochemical process, a scientist isolates and studies the
individual components in vitro, then pieces together the parts to get a coherent picture of the
overall process. To study a particular gene among the tens of thousands of genes nested in the
billions of base-pairs of a genome, techniques for DNA cloning is used, and the very purpose of
Experiment 2 is to clone and isolate a particular V. harveyi chb gene for its further analysis and
research. In Experiment 1, genomic samples from an aquafarm suspected of a SDS outbreak were
analyzed and compared to the Category 1 chb gene by RFLP and Southern Blot analysis. Based
on the results, it was discovered that one of the genomic samples, 092308.C6, is affected by a
unique category of SDS rather than the Category 1 SDS for which a screening kit was
developed. This unique category of SDS is known to have a higher mortality rate and level of
severity than the wildtype SDS outbreak. Therefore, DNA cloning techniques are used to clone
out the V. harveyi chb gene from this genomic mixture to sequence the DNA and test the activity
of protein. The analysis of this particular chb gene through cloning allows determination of any
genetic abnormalities within the gene that are linked to this unique category of SDS.
The basis of molecular DNA cloning involves separating a specific gene or DNA segment
from a larger chromosome, attaching it to small molecule of carrier DNA, and then replicating
this modified DNA thousands or millions of times through both an increase in host cell number
and the creation of multiple copies of the cloned DNA in each cell. The result is selective
amplification of a particular gene or DNA segment so that it is purified essentially to
homogeneity. Thereafter, its structure and function can be comprehensively studied by DNA
sequencing, in vitro expression studies, mutagenesis, etc. There are a variety of cloning methods
used in the field of science as explained in Table 1.

Cloning Methods
Traditional
Cloning
uses restriction endonucleases that recognize and cleave DNA at specific sequences
to generate a set of smaller fragments, which are joined to a suitable cloning vector
by using DNA ligases
Seamless Cloning
allows two or more DNA fragments to be joined precisely so that no unwanted
nucleotides are added at junctions between DNA fragments.

Recombinational
Cloning
a technique based on site-specific recombination that is independent of the insert
DNA sequence to be cloned, enabling rapid and efficient transfer of DNA inserts
into multiple expression systems
Ligation
Independent
Cloning
does not involve in vitro ligation; the annealing of the insert and the vector is
performed in the absence of ligase by simple mixing and incubation of the DNA
fragments
PCR Cloning
Blunt-end cloning is a conventional technique used in cloning PCR-amplified DNA
fragments. In this method, PCR products with blunt ends and blunt-cut vectors are
ligated together. DNA fragments are inserted into the vectors in both orientations,
therefore, directional cloning is not available
Sticky-end cloning uses carefully designed primers that have restriction sites
introduced into the 5 ends. As amplification proceeds, the primers are integrated
into the PCR products. Restriction sites introduced into the two PCR primers can be
identical or different. If they are different. Then the PCR products may be inserted
into the vectors in only one orientation, enabling directional cloning
Table 1: Types of Cloning Methods Available Today
In Experiment 2, a sticky-end cloning method amplifies the chb gene and at the same
time, adds restriction sites Acc665I and SalI to the ends of the amplified DNA, so that it can
be easily cloned into the pUC19 cloning vector. The primers for the sticky-end cloning are
carefully designed to consist of 1) extra base pairs on the 5 end of the primer that assist with
restriction enzyme digestion 2) the chosen restriction enzyme sequence for cloning and 3) the
region of the primer that binds to the sequence to be amplified. These specially designed primers
allow the amplified portion of chb gene product to have a sticky-end overhang including Acc665I
and SalI restriction sites that provides base complementarity to the pUC19 cloning vector after
double digestion reaction is complete. Since overhangs of the double digested insert and vector
have are complementary to each other, ligation and transformation may seem straightforward.
However, there are several problems that must be addressed in order to proceed further in the
cloning process.
1) Partial digestion of the pUC19 is likely to reform an empty pUC19 in the presence
of DNA ligase.
2) The small fragment of the Multiple Cloning Site that was removed from the
pUC19 can relegate back into the pUC19 instead of the chb insert.
3) Contaminating exonucleases can chew away at the ends of the insert and vector,
creating blunt ends. These blunt end fragments are likely to undergo blunt end
ligation, resulting in a plasmid similar to the pUC19, only missing a small portion
of the Multiple Cloning Site.
To address these problems and also reduce the background colonies, Phosphatase treatment, Kill-
cut treatment, solution and gel purification are utilized before going on to the ligation.
Phosphatase treatment minimizes self-ligated vectors by removing the 5 phosphate group from
the digested pUC19. Kill-cut treatment involves digesting the ligation products with a
restriction enzyme that will cut the pUC19 vector multiple cloning site in between the Acc655I
and SalI restriction sites but does not cut the chb insert. The kill-cut treatment will linearize any
plasmid that does not contain the insert of choice. Solution and Gel Purification methods allow
separation of DNA fragments from each other based on their size. Table 2 summarizes the
problems that each treatment addresses.

Type of
Problem
Phosphatase
Treatment
Kill-cut
Treatment
Solution
Purification of
DD vector
Gel Purification
of DD vector
Small and Large
piece of vector
ligate back
together
Removal of the 5
phosphate group of
the vector prevents
the formation of
empty pUC19
The empty pUC19
vector is linearized
by the restriction
enzyme is not
transformed into
bacteria
The large piece of
vector is purified
away from the
small fragment,
preventing
religation
Allows the small
and large
fragments of the
vector to be
separately purified
and isolated
Two pUC19
vectors ligate
Removal of the 5
phosphate group
prevents any
vector fragments
from religating
Not solved Not solved Not solved
Exonuclease
contamination
& blunt end
vector ligation
Absence of the 5
phosphate group
prevents blunt
ends to relegate
Not solved Not solved Not solved
Partial digest of
vector
Removal of the 5
phosphate group of
the vector prevents
the formation of
empty pUC19
The empty pUC19
reformed in the
ligation reaction is
linearized by the
kill-cut treatment
Not solved Not solved
Incomplete
digest of vector
Not solved
The unaffected
pUC19 vector is
prevented from
transforming into
bacteria due to its
linearization after
the kill-cut
treatment
Not solved
The incompletely
digested pUC19
can be cut out and
removed away
from other
fragments present
on the agarose gel
Table 2: Types of Problems in the Cloning Process and Possible Solutions
Once the problems discussed above are addressed and solved, the chb insert and pUC19 vector
can be ligated to form the recombinant plasmid. A ligation reaction that has no chb insert added
is set up as a control. Kill-cut treatment on the ligation reactions linearizes any empty pUC19
plasmids or blunt-end ligated pUC19 plasmids, preventing their transformation. All of the ligated
products are then transformed into bacteria, allowing both selection based on antibiotic resistance
and screening based on X-Gal hydrolysis by -galactosidase. The desired recombinant plasmids
with double digested pUC19 with portion of the chb gene inserted into its multiple cloning site
will grow up as white colonies in transformation.
Reviewing the cloning methods used in the process, one can make a hypothesis that
transformation reactions will work best when the phosphatase treated pUC19 vector is ligated
with the solution purified chb insert to form the recombinant plasmid, which is then treated
with the kill-cut method. The phosphatase treated vector will have been kept from self relegation;
the solution purified chb insert would have been isolated away from any PCR buffer, polymerase
and primers that could possibly inhibit the action of restriction enzymes; the kill-cut treatment
would have minimized background colonies in which the chb insert in absent. Kill-cut treatment
completely blocks the possibility of an empty pUC19 vector transforming into the bacterial cell,
and thus, it is hypothesized reactions using kill-cut treated vector and purified chb insert will lead
to the better results than those using phosphatase treated vectors.

Methods
The experimental methods for the cloning of the unique SDS outbreak V.harveyi chb gene
can be found in the BMB 445W Spring 2014 Laboratory Manual Experiment 2: Clone the V.
harveyi chb gene from an SDS isolate into pUC19 to make a new recombinant plasmid (pBMB2).
In order to provide an overview of the major points in the ligation and transformation steps,
Table 3 is listed below summarizing the characteristic set up of each transformation.

Ligation Sample used
in Transformation
Vector Treatment after Double
Digestion
Addition of chb Insert
A Phosphatase Treatment No
B Phosphatase Treatment Yes
C No Extra Treatment No
D No Extra Treatment Yes
E Kill Cut after Ligation Yes
Table 3: Summary of the Transformation Reaction Setups
Results and Discussion
The transformed bacteria plated on the LB-AMP-X-gal-IPTG plates were incubated at
37C to allow for colony growth. The number of blue colonies, white colonies, and pale blue
colonies on each plate were recorded for each group and collected as a section. The pale blue
colonies implied 1) reduced expression of the omega fragment due to a particular disruption in
the gene, or 2) white colonies with bacteria expressing an unknown factor that nonspecifically
hydrolyzed the X-gal. In either possibility, the presence of a blue color indicated the possibility
of hydrolytic ability that breaks down X-gal, and thus in this section, pale blue was not
considered as a desired white colony.
(All of the Charts and Tables of the Results and Discussion Section are found on the Data
Chart Sheet attached to the back this report)
Table 4 shows the recorded number of colonies as an average of the section, and the
individual record collected from my lab group. The total number of colonies show in both
Section Average and Individual results that Transformation Reactions C and D the two
reactions that used vectors with no extra treatment supported largest number of bacterial
colonies on the transformation plate. The total number of colonies, however, is not the
determinant for the success of transformation. This can be seem more clearly in Graph 1, in
which the numerical values from Table 4 are graphed as comparative column bars. While
Transformation Reactions C and D do have the most colonies, they also have a highest number of
blue colonies as opposed to the desired white colonies. This indicates that the untreated vectors
produced a higher number of background colonies in which the chb insert is absent. Comparing
the graph bars for Transformations A and B against Transformation C and D, it is observed that
the roughly estimated ratio of blue colonies to white colonies is lower in the phosphatase treated
A and B reactions while higher in the non-treated C and D reactions; the ratio was as low as
3.24:1 in reaction B and as high as 16.65:1 in reaction C. Therefore the comparison of the total
number of colonies with component blue and white colonies suggests that phosphatase treatment
does indeed serve its purpose in reducing the background by preventing the pUC19 from
religating with its self-components. Yet, the phosphatase treatment also seemed to reduce the
total amount of colonies growing on the plate, which can be an inhibiting factor of the
transformation reaction. Transformation reaction A exhibits this problem: both the section
average and individual results result in very few or no white colonies grown, resulting in loss of
data to analyze for positive products.
There is another important factor to consider when looking at the number of colonies: the
presence of the chb gene insert. Whether or not the chb gene insert was added to the ligation
reaction seemed to have a rather significant effect on the number of white colonies. This is
demonstrated in Graph 2. The percentage of only the white colonies in each transformation
reaction is graphed linearly. The section average data indicated in blue show a strong trend; while
it was expected that the transformation reactions that used phosphatase treated vectors would
produce a notably higher number of white colonies, the actual percentage of white colonies
between transformation reactions using phosphatase treated vector (A and B) and those using
non-treated vectors (C and D) were strikingly similar in value. As mentioned above, ligation
reactions A and C are set up as controls so that no chb gene is added to the, and as a result the
expected result is a low percentage of white colonies due to the insert being the rate-limiting
factor. However, the expected hypothesis that phosphatase treatment would reduce the
background blue colonies and increase the white colonies is not met in the results. In the class
average data, reaction B displays 23.6% of white colonies and reaction D displays 23.5% of
white colonies, almost identical to reaction B. This indicates that while the colony count numbers
from Graph 1 may seem to show an overall reduced background in reactions using phosphatase
treated vectors, the actual percentage of white colonies support that phosphatase treatment in the
experimental procedure did not work well. This is further substantiated by the individual data
(indicated in red) that shows a similar trend to the section average trend except for the reduced
percentage of white colonies in reaction E.
When it comes to the kill-cut treatment, the data supports that this treatment is effective in
background reduction. The numerical values total colonies as well as each of blue and white
colonies in reaction E in Graph 1 are significantly lower than reactions B, C, and D, which
produced total number of colonies as high as 928. However, the percentage of white colonies is
the highest at 46% in Graph 2 (Section Average Data). The individual data displays a slightly
lower percentage of white for reaction E, but this decrease could be contributed to experimental
error in carrying out the heat-shock and chilling phases; cloning and transformation can be very
sensitive in that even with the identical experimental procedures, results vary due to minor
differences that may arise in carrying out the experiment. Another reason for the decrease in
white colonies could arise during plating: the written procedure instructed to spin down the
transformation reactions and remove majority of the media but 100 ul. This process of removing
the media was not measured in a quantitatively accurate way and thus, the amount of cells
transferred to the plate could have been reduced. Also, there is the probability that some cells
were killed due to the heat of the glass hockey-stick.
When looking at just the colony counts, reaction D seems the most desirable for both the
section average data and individual data, for having produced the highest number of white
colonies. Although white colonies indicate positive results, they must be tested in order to
verify that the bacteria do in fact contain the recombinant DNA of interest and not a nonspecific
plasmid that would confer antibiotic resistance. Therefore, colony PCR was performed using
primers that amplify a portion of the chb gene, allowing results to confirm the presence of the
insert within the pUC19 vector. For the negative and positive controls of the PCR, bacterial
transformation using pUC19 and bacterial transformation with pBMB was used, respectively.
The empty pUC19 does not contain a chb gene insert and thus does not produce any PCR bands,
while the pBMB produces a prominent band due to the fact that it carries the chb gene within its
plasmid. The colony PCR results are shown in Graph 3. Looking at the number of positive
colonies for the section average, reaction E seems to be the most desirable, while the individual
data suggests that reaction B proves desirable with its number of strong positives. Overall, the
higher number of negative colonies supports that this cloning process is a complicated procedure
with a larger margin for error. It also indicates that there are a large number of false positives
within the white colonies. Factors such as mutations and non-specific PCR products or pieces of
genomic DNA being incorporated into the pUC19 vector can produce false positives. Most
importantly, exonuclease contamination can chew away sticky ends on pUC19 and allow for
blunt end ligation. This product of blunt-end ligation is able to be incorporated in to the bacterial
cell, but has a disturbed multiple cloning site that blocks its X-gal hydrolysis by -galactosidase.
Reactions F and G were set up as the control reactions for the transformation. No DNA
was used for the transformation reaction F, so it was expected to show no growth on the plate,
which the result follows. Reaction G used either the undigested pUC19 or undigested pBMB as
the plasmid for incorporation to show that the transformation reaction is set up appropriately and
that the bacterial cells do actually show competence with the ability to take up plasmid. The
positive control was expected to show numerous colonies, as supported by the results.
Combining the data from Table 4 and Graphs 1,2,3, it can be supported that that reaction
E, with the highest overall % white colonies and overall number of strong positives, yields the
best results in this cloning experiment. The colony count alone seems to show that reaction E
produced a significantly smaller number of white colonies compared to other reactions, but the
colony PCR suggests that even with a small amount of white colonies reaction E has very little
false positives. Transformation reactions C and D on the other hand seem to have a larger
proportion of false positives, evidenced by the lack of product bands after the PCR reaction. This
supports my initial hypothesis that transformation reactions using kill-cut treated vectors and
purified chb gene inserts will lead to the best cloning results.

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