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Hijack Module (Fast robust oscillator) Notebook

25th June 2015

DH5α competent cells were transformed with the following biobricks:

BioBrick

Part

Backbone

Kit plate, Well

BBa_C0080

araC protein coding sequence

pSB1C3

3,11B

BBa_C0012

LacI protein coding sequence

pSB1C3

pSB1C3

 

The plates were kept at 37°C for 14 hours.

 

26th June 2015

Transformation results:

Biobrick

Number of colonies

BBa_C0080

0

BBa_C0012

3

 

As number of colonies obtained was very low, the transformation of these biobricks was repeated.

 

27th June 2015

Transformation results:

Biobrick

Number of colonies

BBa_C0080

3

BBa_C0012

60

 

Inoculation:

A colony from BBa_C0080 plate was inoculated in 7ml LB media + 7μL Chloramphenicol. Same was done for BBa_C0012. These cultures were kept at 37°C for 12 hours.

28th June 2015

No growth was observed in both the liquid cultures.

A new colony was inoculated again.

29th June 2015

Growth observed in the cultures of BBa_C0080 and BBa_C0012. Gylcerol stocks were made for both.

Plasmid isolation was done using Alkaline Lysis protocol for the 2 cultures. (1.5ml culture x 3)

Concentrations obtained from nanodrop:

Sample

Concentration (ng/μL)

A260/A280

BBa_C0080 (1)

3301.8

2.02

BBa_C0080 (2)

3670.9

1.98

BBa_C0080 (3)

2777.6

1.95

BBa_C0012 (1)

4560.0

1.97

BBa_C0012 (2)

4542.1

1.94

BBa_C0012 (3)

5123.5

1.61

 

A single digest was set for one of each of these plasmids:

(all volumes in μL)

BBa_C0012

BBa_C0080

PstI

0.3

0.3

10x NEBuffer 3

1

1

100x BSA

0.1

0.1

DNA

0.5

0.5

MilliQ H20

8.1

8.1

Total Volume

10

10

 

-          Kept at 37°C for 2 hours

-          Heat Inactivation: 80°C for 20 minutes

 

Gel Electrophoresis:

-          Digested samples run on a 1% gel.

-          Voltage: 100V

-          Time run: 50 minutes

 

Both samples showed an RNA smear. This occurred as the Alkaline Lysis solution I did not have RNAse. After observing this smear, RNAse was added to the solution.

Re-inoculation of the two cultures was done

 

 

1st July 2015

A plasmid isolation using Alkaline lysis was done.

Sample

Concentration (ng/μL)

A260/A280

BBa_C0080 (1)

4548.1

1.98

BBa_C0080 (2)

3411.9

2.01

BBa_C0080 (3)

3492.6

1.98

BBa_C0012 (1)

2258.4

1.99

BBa_C0012 (2)

2442.6

2.00

BBa_C0012 (3)

2691.4

1.97

 

A digest was set up with a sample of each plasmid and then run on a gel.

The gel showed bands at correct length, but the band intensity was extremely low and did not reflect the concentration obtained on nanodrop. A new method for plasmid isolation was needed.

2nd July 2015

BBa_K094120 arrived from iGEM Headquarters and was inoculated in 5ml LB media + 5μL Ampicillin. Kept at 37°C for 12 hours.

 

3rd July 2015

The liquid culture of BBa_K094120 was plated and kept overnight at 37°C

5th July 2015

A single colony was inoculated for BBa_K094120. Kept at 37°C for 12 hours

6th July 2015

Plasmid isolation of BBa_K094120 using Alkaline Lysis:

Sample

Concentration (ng/μL)

A260/A280

BBa_K094120 (1)

1050.8

1.88

BBa_K094120(2)

1493.7

1.95

BBa_K094120 (3)

1365.2

1.97

 

29th July 2015

1% gel run with all samples for the 3 plasmids BBa_C0080, BBa_C0012, BBa_K094120.

All bands seen had low intensity which did not correlate to the concentration on nanodrop.

 

11th August 2015

Revive glycerol stocks for BBa_C0080, BBa_K094120 and BBa_C0012. A 50 ml culture was inoculated for BBa_C0080 and BBa_C0012 for a Midiprep using Qiagen kit.

Kept at 37°C overnight

 

12th August 2015

Midiprep using Qiagen Kit results for BBa_C0080 and BBa_C0012:

Sample

Concentration (ng/μL)

A260/A280

BBa_C0012

7.0

3.62

BBa_C0080

27.8

2.07

 

Since results obtained were very low, a new “Qiagen spin miniprep” kit was obtained.

 

17th August 2015

Primary cultures used to make an overnight 5ml culture of BBa_C0080, BBa_C0012 and BBa_K094120.

18th August 2015

Qiagen Spin Miniprep protocol followed. Results:

Sample

Concentration (ng/μL)

A260/A280

BBa_K094120

250.7

1.91

BBa_C0080

165.5

1.92

BBa_C0012

195.4

1.92

 

A digestion was set to check plasmid length:

(all volumes in μL)

BBa_C0080

BBa_C0012

BBa_K094120

DNA

2

2

2

EcoRI

0.2

0.2

0.2

10x EcoRI Buffer

1

1

1

100x BSA

0.1

0.1

0.1

MilliQ H20

6.4

6.4

6.4

Total Volume

10

10

10

 

Gel: 1% gel run at 100V for 40 minutes.

The bands obtained for all 3 plasmids were at the correct length. The uncut plasmid ran faster than cut plasmid.

21st August 2015

Inoculated BBa_I13504 (GFP, used as reporter in oscillator construct) from previously transformed plate. Kept at 37°C for 12 hours

22nd August 2015

-          Miniprep of BBa_I13504:

Sample

Concentration (ng/μL)

A260/A280

BBa_I13504

122.4

1.94

 

-          Transformation of the biobrick BBa_B0034 (RBS) in DH5α competent cells (Ampicillin antibiotic)

-          Plates kept at 37°C for 14 hours

 

23rd August 2015

Inoculated a colony for BBa_B0034 and kept at 37°C for 12 hours.

24th August 2015

Plasmid Isolation using Qiagen Miniprep kit:

Sample

Concentration (ng/μL)

A260/A280

BBa_B0034

95.2

1.95

 

25th August 2015

-          Transformation of the biobrick BBa_B0015 (double terminator) in DH5α competent cells (Chloramphenicol antibiotic)

-          Plates kept at 37°C for 14 hours

26th August 2015

Transformation Result:

Number of colonies for BBa_B0015 = 8

-          Inoculated BBa_B0015 in 5ml LB media + 5 μL Chloramphenicol

-          Kept at 37°C for 12 hours

28th August 2015

Plasmid Isolation of BBa_B0015 using Qiagen Miniprep kit:

Sample

Concentration (ng/μL)

A260/A280

BBa_B0034

72.3

1.95

 

29th August 2015

Single Digest to check plasmid length:

(all volumes in μL)

BBa_B0015

BBa_I13504

BBa_B0034

DNA

4

2

2

EcoRI

0.2

0.2

0.2

10x EcoRI Buffer

1

1

1

100x BSA

0.1

0.1

0.1

MilliQ H20

5.7

5.7

5.7

Total Volume

10

10

10

 

Gel: 1% gel run with uncut and cut samples at 100V for 40 minutes.

The 3 single cut bands were at the right length.

 

6th September 2015

Digestion for 3A assembly of constructs of the oscillator construct:

(all volumes given are in μL)

BBa_

K094120

 

BBa_

C0080

BBa_

C0012

BBa_

I13504

BBa_

B0034

BBa_

B0015

Ampicillin Backbone

Chloramphenicol Backbone

Kanamycin Backbone

DNA

3

3

3

3

4

5

10

10

10

10x NEBuffer 2

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

BSA

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

EcoRI

0.5

0.5

0.5

-

-

-

0.5

0.5

0.5

XbaI

-

-

-

0.5

0.5

0.5

-

-

-

SpeI

0.5

0.5

0.5

-

-

-

-

-

-

PstI

-

-

-

0.5

0.5

0.5

0.5

0.5

0.5

H20

13

13

13

13

12

11

6

6

6

Total

20

20

20

20

20

20

20

20

20

-          Kept at 37°C for 5 hours

-          Heat Inactivation at 80°C for 20 minutes

-          The following devices were to be assembled:

-          BBa_K094120 + BBa_B0034 + Chloramphenicol backbone = Device 2a

-          BBa_C0080 + BBa_B0015 + Kanamycin backbone = Device 2b

-          BBa_C0012 + + BBa_B0015 + Ampicillin backbone = Device 2c

-          BBa_K094120 + BBa_I13504 + Kanamycin backbone = Device 2d

 

 

 

Ligation:

(all volumes in μL)

Device 2a

Device 2b

Device 2c

Device 2d

Ampicillin Backbone

-

-

2

-

Chloramphenicol Backbone

2

-

-

-

Kanamycin Backbone

-

2

-

2

BBa_K094120

2

-

-

2

BBa_B0034

2

-

-

-

BBa_C0080

-

2

-

-

BBa_B0015

-

2

2

-

BBa_C0012

-

-

2

-

BBa_I13504

-

-

-

2

T4 DNA ligase

0.5

0.5

0.5

0.5

Ligase Buffer

1

1

1

1

MilliQ H20

2.5

2.5

2.5

2.5

Total Volume

20

20

20

20

 

Ligation kept overnight at room temperature.

 

7th September 2015

-          Transformation of the ligation mix into DH5α cells

-          Kept at 37°C overnight

 

8th September 2015

Transformation results:

Ampicillin Negative Control : Lawn growth

Kanamycin Negative Control : Zero colonies

 

Plate

Number of Colonies

2a

10

2b

12

2c

Lawn growth

2d

5

 

Ampicillin degraded. 2c re-transformed

Inoculation of a single colony from each plate in 5ml LB media + 5μL antibiotic

Kept at 37°C for 12 hours

9th September 2015

Miniprep by Qiagen Kit:

Sample

Concentration (ng/μL)

A260/A280

2a

283.4

1.91

2b

122.3

2.09

2d

226.6

1.96

 

Single Digest with EcoRI to check plasmid length:

Backbone-Backbone ligation was observed on the gel.

A new ligation was setup. This time insert:vector ratio was increased. Kept at 37°C for 2 hours.

These ligation mix were transformed into Dh5α competent cells.

 

10th September 2015

Growth observed in negative control plates.

An overnight digestion was set for the individual plasmids again. Kept at 37°C for 12 hours

 

11th September 2015

Ligation set for 2 hours and then transformed.

12th September 2015

Colonies of device 2a, 2b and 2c obtained. No colonies on 2d.

3 colonies from each plate were inoculated, incubated for 12 hours at 37°C and miniprepped.

These were then digested with EcoRI to check the length of the plasmid on a gel.

The bands were as expected.

An overnight digest followed by 2 hour ligation was set to assemble to following device:

Device

Part1

Part2

Backbone

5a

2a

2b

pSB1A3

5b

2a

2c

pSB1K3

 

Two samples of 5b were assembled – one low copy and second high copy backbone

BBa_I13504 in Ampicillin backbone was chosen to assemble device 2d. This biobrick was digested and ligated.

Devices 5a, 5b and 2d were transformed.

13th Spetember 2015

Transformation Results:
                Negative Control: Zero

Plate

Number of Colonies

5a

15

5b (low copy)

70

5b (high copy)

26

2d

10

 

Three colonies were inoculated from each plate, incubated at 37°C and then miniprepped.

These were then single digested using EcorI to check on gel.

2d, 5a and 5b high copy showed correct bands.

 

18th September 2015

2d construct was characterized by induction with different concentrations of IPTG and arabinose.

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Chromoproteins

8th June, 2015

 

●     The following biobrick parts were revived from the iGEM 2015 distribution plates using the iGEM protocol Standard protocol.

●     Transformation was carried with Ultra-competent cells for DH5α strain of E.coli (with 200µL of LB).

●     200µL of the revived product was plated on LA+antibiotic.

●     The plates were kept at 37℃ overnight (12-16 hours).

 

Part Number

Location in Kit

Backbone

J23110 (Constitutive Promoter)

Plate 4, Well 19D

Ampicillin

K592025 (RBS+amilCP)

Plate 1, Well 19C

Chloramphenicol

J04450 (RFP)

Plate 4, Well 2H

Ampicillin

 

 

Negative Control: For both antibiotics (Amp and Cam)

Positive Control: RFP

 

9th June, 2015

 

 

Part Number

Number of colonies

J23110 (Promoter)

Many colonies

K592025 (RBS+amilCP)

Many single colonies

J04450 (RFP)

Lawn growth

Negative Control

No colonies

 

●     Next, the colonies were inoculated in 5mL LB+ 0.1% antibiotic.

●     They were kept in an incubator at 37℃ overnight (12-16 hours) at around 180-200 rpm.

 

10th June, 2015

 

●     Growth was observed in K592025, but no growth in J23110.

●     Again a colony was picked from the plate with J23110 and inoculated in 5mL LB+ 0.1% antibiotic.

●     They were kept in an incubator at 37℃ overnight (12-16 hours) at around 180-200 rpm.

 

11th June, 2015

 

●     Again no growth was observed in J23110 (even after inoculating twice).

●     Transformation was done again for J23110

●     Different promoter was also transformed.

 

 

Part Number

Location in Kit

Backbone

J23119 (Constitutive Promoter)

Plate 3, Well 17O

Chloramphenicol

 

13th June, 2015

 

●     The two promoters were tranformed with 200µL SOC media which was prepared using the iGEM protocol (earlier transformations with LB).

●     200µL of the revived product was plated on LA+antibiotic.

●     The plates were kept at 37℃ overnight (12-16 hours).

 

14th June, 2015

 

 

Part Number

Number of colonies

J23110 (Promoter 1)

30

K592025 (RBS+amilCP)

3

J23119 (Promoter 2)

47

Negative Control

No colonies

 

 

●     The colonies obtained were inoculated in 5mL LB+ 0.1% antibiotic.

●     They were kept in an incubator at 37℃ overnight (12-16 hours) at around 180-200 rpm.

 

18th June, 2015

 

Plasmid isolation was carried out using the Alkaline Lysis Protocol from Sambrook and Maniatis.

Resuspended in TE Buffer.

 

 

 

 

Results of Nanodrop:

 

Sample

Vial Number

A260/A280

Concentration (ng/µL)

K592025

1

1.99

6691.9

 

2

2.11

4749.5

 

3

2.12

4158.3

J23119

1

2.10

4865.3

 

2

2.09

2757.4

 

3

2.10

4902.3

J23110

1

2.09

5121.8

 

2

2.15

8148.0

 

3

2.13

3223.8

 

●     The alkaline lysis buffer I wasn’t added with RNase.

●     The plasmid isolation was repeated with RNase added to Buffer I.

 

 

27th June, 2015

 

●     The colonies were again inoculated in 5mL LB+ 0.1% antibiotic.

●     They were kept in an incubator at 37℃ overnight (12-16 hours) at around 180-200 rpm.

 

 

28th June, 2015

 

●     RNase (10mg/mL stock) was added to Alkaline Lysis Buffer I.

●     Resuspension in milliQ water.

 

Results of Nanodrop:

 

Sample

Vial Number

A260/A280

Concentration (ng/µL)

K592025

1

2.13

56.9

 

2

1.39

67.4

 

3

2.06

58.5

J23119

1

2.16

34.1

 

2

2.15

32.3

 

3

1.39

116.1

J23110

1

2.08

158.4

 

2

2.02

3222.1

 

3

1.95

580.4

 

 

29th June, 2015

 

●     The colonies were again inoculated in 5mL LB+ 0.1% antibiotic.

●     They were kept in an incubator at 37℃ overnight (12-16 hours) at around 180-200 rpm.

 

30th June-10th July, 2015

Again, alkaline lysis was done.

 

Results of Nanodrop:

 

Date

Sample

Vial Number

A260/A280

Concentration (ng/µL)

30/6

J23119

1

1.94

2376.8

30/6

J23119

2

1.95

2301.2

30/6

J23119

3

1.88

1662.9

8/7

J23119

1

1.99

1616.5

8/7

J23110

2

1.98

618.9

10/7

K592025

1

1.96

1641.1

10/7

K592025

2

1.97

1457.0

10/7

K592025

3

1.99

1404.6

 

 

 

 

 

 

 

 

 

 

26th August and 3rd September

 

Results for Nanodrop by Qiagen spin mini kit

 

Sample

A260/A280

Concentration (ng/μL)

J23119

2.04

121.1

J23119

1.89

329.3

K592029

1.96

158

K592029

1.88

407.3

 

 

 

 

 

 

26th August

Single digestion reaction

 

(in μL)

K592025

J23119

EcoRI

0.3

0.3

DNA

2

2

10XEcoRI Buffer

1

1

100X BSA

0.1

0.1

Distilled water

6.6

6.6

Total Volume

10

10

 

 

27th August

 

3A assembly double digestion

(in μL)

Promoter(J23119)

amilCP(K592025)

Linear backbone

DNA

2

2

10

10X Buffer2

2.5

2.5

2.5

100X BSA

0.5

0.5

0.5

EcoRI

0.5

-

0.5

SpeI

0.5

-

-

PstI

-

0.5

0.5

XbaI

-

0.5

-

DpnI

-

-

0.5

Distlled water

14

14

5.5

Total Volume

20

20

20

Kept at 37°C for 4 hours,  heat-kill at 80°C for 20min.

Colonies obtained (~10),  were inoculated and miniprep was done with qiagen kit.

 

Table3: Double digestion for 2A assembly

2A assembly Double digestion:

 

(in μL)

Promoter(J23119)

amilCP(K592025)

DNA

7.8

7

10X Buffer2

1

1

100X BSA

0.2

0.2

SpeI

0.5

0.5

PstI

0.5

-

XbaI

-

0.5

Distilled water

0

0.8

Total Volume

10

10

 

 

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Detection Enzyme Substrate Notebook

DETECTION: Enzyme substrate reaction

Parts:

Biobrick

Part

Size(bp)

Location

1

BBa_K1509003

Constitutive promoter

2129

Ordered from iGEM HQ

2

BBa_K823016

RBS + full length LacZ

5163

Kit plate 2

5/08/15

Transformation of BBa_K823016 (LacZ gene along RBS) plasmid in DH5α competent cells

Protocol: iGEM Transformation protocol

Master plate is made

6/08/15

Observed colonies on the plate

12/08/15

Liquid culture is prepared for BBa_K823016

Protocol

  • 5ml of LB in 15ml falcon
  • Add 5µl of cam
  • Pick a colony from the plate and dip in falcon
  • Incubate for 12-14hrs

22/08/15

BBa_K1509003 (promoter) is ordered and got from iGEM HQ. NEB10 is the cell line

Preparation of liquid culture

  • 5ml LB in 15ml falcon
  • Add 5µl of cam
  • A loop of nichrome dipped in NEB10 cells containing promoter plasmid
  • Incubate for 12-14hrs

23/08/15

Streaking of plate

  • Prepare a cam media plate of 20ml LA
  • Streak the plate using previously prepared liquid culture
  • Overnight Incubation

24/08/15

Grow liquid stalk culture

  • 5ml LB in 15ml falcon
  • Add 5µl of cam
  • Pick a colony from master plate and dip in media
  • Overnight incubation

Preparation of glycerol stalk

  • 0.5ml 80% glycerol in cryotube
  • 0.5ml bacterial culture
  • Flash freeze it using liquid nitrogen

24/08/15

Plasmid isolation by alkaline lysis

Chemicals required:

  1. CAM antibiotic
  2. LB media
  3. Alkaline lysis buffer 1
  4. Alkaline lysis buffer 2
  5. Alkaline lysis buffer 3
  6. Ethanol
  7. Phenol: Chloroform (1:1)
  8. STE
  9. TE with RNase A

Protocol

  1. Inoculate 5ml of rich media containing the appropriate antibiotic (CAM)
  2. Incubate culture overnight at 37 deg
  3. Pour 5ml of culture into a microfuge tube
  4. Centrifuge in microfuge for 3 min at 4deg
  5. Remove media by aspiration leaving bacterial pellet as dry as possible
  6. Resuspend the pellet in 100microl of ice-cold alkaline lysis solution 1 by vortexing
  7. Add 200microl of alkaline lysis solution 2 , mix with inverting
  8. Add 150 microl ice-cold alkaline lysis solution 3 mix with invertexing
  9. Store tube on ice for 3-5 min
  10. Centrifuge bacterial lysate ,5 min 13000rpm at 4deg in microfuge
  11. Transfer supernatant to fresh tube
  12. Add equal volume of phenol: chloroform
  13. Mix the organic and aqueous phase by vortexing
  14. Centrifuge at maximum speed 2 min at 4deg
  15. Transfer the aqueous upper layer to fresh tube
  16. Add 2 volume of ethanol
  17. Mix by vortexing for 2 min
  18. Centrifuge for 5min at 4deg
  19. Remove supernatant by aspiration
  20. Remove all liquid keeping inverted position
  21. Add 1ml of ethanol to pellet
  22. Centrifuge for 2min at 4deg
  23. Remove all supernatant by aspiration
  24. Remove ethanol beads and evaporate it at room temperature
  25. Dissolve nucleic acid in 50microl of TE containing RNases A
  26. Vortex the solution gently for 2 sec
  27. Store DNA solution at -20deg

Result: no DNA pellet after precipitation by ethanol

Comment: Most probably step 12 is done in incorrect way

Inoculation of a colony of BBa_K823016 and BBa_K1509003

25/08/15

Plasmid isolation of BBa_K1509003 using QIAGEN miniprep kit

Protocol: QIAGEN Miniprep gravity column is used

Result: No DNA after precipitation by isopropanol addition

26/08/15

Inoculation of BBa_K823016 and BBa_K1509003

Plasmid isolation using QIAGEN Miniprep spin column

Protocol: QIAGEN Miniprep spin column protocol is used

Results:

part

Concentration(ng/µl)

BBa_K823016

 52.2

BBa_K1509003

38.7

Inoculation of BBa_K823016 and BBa_K1509003 for alkaline lysis

27/08/15

Plasmid isolation using Alkaline lysis Miniprep in AB Lab

Chemicals Required: Alkaline lysis buffer 1, 2, 3 buffer 2 freshly prepared, RNases

Protocol: standard protocol is used from sambrook molecular cloning book as mentioned earlier

Result

part

Concentration(ng/µl)

BBa_K823016

2650.8

BBa_K1509003

4080.3

This has high RNA contamination

Gel Electrophoresis is done which shows high contamination of RNA

2/09/15

Inoculation of both BBa_K823016 and BBa_K1509003

Plasmid isolation using QIAGEN Miniprep spin column

Protocol: QIAGEN Miniprep spin column protocol is used

Result:

           

part

Concentration(ng/µl)

BBa_K823016

 275.2

BBa_K1509003

87.3

Gel Electrophoresis is done to check isolated plasmid

8/09/15

Inoculation of BBa_K1509003 in 5ml LB and 5µl cam

9/09/15

Gel Electrophoresis (1%)

Chemicals required: 0.5gm agarose, 50ml TAE, 5µl EtBr

Inoculation of both BBa_K823016 and BBa_K1509003 in 5ml LB each

10/09/15

Plasmid isolation using QIAGEN Miniprep spin column

Protocol: QIAGEN Miniprep spin column protocol is used

Result:

           

part

Concentration(ng/µl)

BBa_K823016

 735

BBa_K1509003

234

Single cut digestion

BBa_K823016: 1µl

BBa_K1509003:3µl

BBa_K823016 (µl)

BBa_K1509003 (µl)

DNA

1

3

ECoR1 buffer

1

1

ECoR1 enzyme

0.5

0.5

dH2O

7.5

5.5

Total

10

10

Gel Electrophoresis: Fig1

12/09/15

 Fig2

Confirmation of plasmid isolated

                                                                                                                                 

12/09/15

Digestion for 3A assembly

pSB1K3 which is a Kan backbone  used for 3A assembly

BBa_K823016 (µl)

BBa_K1509003 (µl)

1

NEB buffer 2

5

5

2

BSA

0.5

0.5

3

EcoR1-HF

0.5

0.5

4

Spel

0.5

0.5

5

dH20

18.5

18.5

6

Total

20

20

Protocol: standard iGEM 3A assembly protocol is used

13/09/15

Ligation using 3A assembly of biobricks

Protocol: standard iGEM 3A assembly protocol is used

15/09/15

Transformation of ligated parts

Protocol: IGEM transformation protocol is used

Result: No colony is observed on both KAN media plates

Comment: most probably failure of digestion

16/09/15

Inoculation of both BBa_K823016 and BBa_K1509003

Plasmid isolation using QIAGEN Miniprep spin column

Protocol: QIAGEN Miniprep spin column protocol is used

Result:

           

part

Concentration(ng/µl)

BBa_K823016

 102.3

BBa_K1509003

65.5

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