Team:University College London/LabBook/Week15

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== Tuesday (18.09.12) ==
== Tuesday (18.09.12) ==

Revision as of 02:42, 27 September 2012

Contents

Monday (17.09.12)

Aim - We want to check if the ligations have been successfulccessful


Step 1 We carried analytical digest of the following ligations:


  • BBa_K729004 + BBa_J23119-BBa_B0034
  • BBa_K729003 + BBa_J23119-BBa_B0034
  • BBa_K118011 + BBA_I746916


Methods:

Restriction Enzyme Digest Protocol 1

Step 1 - Thawing cells: Thaw all materials on ice

Step 2 - Adding Ingredient: Add the following ingredients to autoclaved/sterile eppendorf tubes

Component Amount (ul) (one enzyme used) Amount (ul) (two enzymes used)
dH20 2.5 1.5
Buffer 1x 1 1
DNA template 5 5
BSA 0.5 0.5
Enzyme 1 1 2
Enzyme 2 N/A 1


Step 3 - Addition of BioBrick: Flick contents gently and centrifuge.

Step 4 - Centrifuge:

RPM: 14000

Time: 1 minute

Temperature: 18oC

Step 5 - Digest Program: Place the samples on a thermocycler under the following conditions:

RPM: 550

Time: 2.5 hours

Temperature: 37oC

Step 6 - Denaturing Enzymes: If you are not running the samples on a gel immediately, denature the restriction enzymes by running the samples on a thermocycler under the following conditions:

RPM: 550

Time: 25 minutes

Temperature: 65oC



Step 2- We run a gel of the digests

Methods:

Electrophoresis Protocol

Preparing the Gel

Step 1: Within a conical flask, add 3ml 50X TAE, 1.5g Agarose, and 150ml RO water

Step 2: Heat for 1 min in microwave. Swirl. Heat again for 30s. If solution is clear stop. Else repeat.

Step 3: Cool solution under running cold water.

Step 4: Add 20ul ethidium bromide (normal concentration of EB solution is 500ug/ul)

Step 5: Pour into a sealed casting tray in a slow steady stream, ensuring there are no bubbles

Running a gel

Step 6: Add 1 part loading buffer to five parts of loading sample

Step 7: Position the gel in the tank and add TAE buffer, enough to cover the gel by several mm

Step 8: Add 5ul of DNA ladder to lane 1

Step 9: Add samples to the remaining wells

Step 10: Run at 100 volts for 1hour and 15 minutes

Imaging the Gel

Step 11: Place gel in GelDoc 2000 chamber

Step 12: Turn GelDoc 2000 chamber on 

Step 13: From computer: Quantity One > Scanner > Gel_Doc_Xr>Manuqal Acquire

Step 14: Alter the exposure/settings to give a clear image.

TAE - Tris-acetate-EDTA

EDTA - ethylenediamine tetraacetic acid



Results:


Gel 1 table 1week 15.png


Conclusion: The bands that we obtained on the gel did not correspond to the sizes we were expecting. For most of the samples we could clearly see a band at around 2000 which most probably is the backbone. These confusing results may be due to mistakes during the ligation or the enzyme digestion. Contamination is another possible reason for these results.



Gel 1 table2week15.png

Tuesday (18.09.12)

Aim - We want to check if the ligations have been successfulccessful


Step 1 We carried analytical digest of the following ligations:


  • BBa_K729003+(BBa_J23119-BBa_B0034)
  • BBa_K118011+BBa_I712069
  • BBa_K729005+BBa_I712069
  • BBa_I746909+K729007


Methods

Restriction Enzyme Digest Protocol 1

Step 1 - Thawing cells: Thaw all materials on ice

Step 2 - Adding Ingredient: Add the following ingredients to autoclaved/sterile eppendorf tubes

Component Amount (ul) (one enzyme used) Amount (ul) (two enzymes used)
dH20 2.5 1.5
Buffer 1x 1 1
DNA template 5 5
BSA 0.5 0.5
Enzyme 1 1 2
Enzyme 2 N/A 1


Step 3 - Addition of BioBrick: Flick contents gently and centrifuge.

Step 4 - Centrifuge:

RPM: 14000

Time: 1 minute

Temperature: 18oC

Step 5 - Digest Program: Place the samples on a thermocycler under the following conditions:

RPM: 550

Time: 2.5 hours

Temperature: 37oC

Step 6 - Denaturing Enzymes: If you are not running the samples on a gel immediately, denature the restriction enzymes by running the samples on a thermocycler under the following conditions:

RPM: 550

Time: 25 minutes

Temperature: 65oC



Step 2- We run a gel of the digests

Methods

Electrophoresis Protocol

Preparing the Gel

Step 1: Within a conical flask, add 3ml 50X TAE, 1.5g Agarose, and 150ml RO water

Step 2: Heat for 1 min in microwave. Swirl. Heat again for 30s. If solution is clear stop. Else repeat.

Step 3: Cool solution under running cold water.

Step 4: Add 20ul ethidium bromide (normal concentration of EB solution is 500ug/ul)

Step 5: Pour into a sealed casting tray in a slow steady stream, ensuring there are no bubbles

Running a gel

Step 6: Add 1 part loading buffer to five parts of loading sample

Step 7: Position the gel in the tank and add TAE buffer, enough to cover the gel by several mm

Step 8: Add 5ul of DNA ladder to lane 1

Step 9: Add samples to the remaining wells

Step 10: Run at 100 volts for 1hour and 15 minutes

Imaging the Gel

Step 11: Place gel in GelDoc 2000 chamber

Step 12: Turn GelDoc 2000 chamber on 

Step 13: From computer: Quantity One > Scanner > Gel_Doc_Xr>Manuqal Acquire

Step 14: Alter the exposure/settings to give a clear image.

TAE - Tris-acetate-EDTA

EDTA - ethylenediamine tetraacetic acid


Gel2table1week15.png


Gel2table2week15.png

Conclusion: We have obtained the expected sizes on for the pcstA (BBa_K118011) +RBS-T7RNAP (BBa_I712069) ligation.


Tuesday (18.09.12)

Picking Colonies
Step 1 – Creating culture media: In a sterile environment, set up X numbers of falcons, each with 5mls of media.


Step 2 - Inoculating Colonies into a Selective Broth:: Add Yul of antibiotic to reach desired antibiotic concentration.


(For Ampicillin this is 50ug/ml, For Kanamycin it is 25ug/ml, for Tetracycline it is 15ug/ml, and for Chloramphenicol it is 25ug/ml)


Step 4 – Selecting a Colony: Select a clear, isolated colony and using an inoculation hoop scoop up a colony onto the tip. Deposit in the falcon tube


Step 5 - Culture: Culture your falcon tubes overnight at a temperature of 37 oC. Leave for no longer than 16 hours.

Tuesday (18.09.12)

Aim – We would like to ligate BBa_I746909 to BBa_K729007 to obtain the following construct pCstA+RBS+T7RNAP+pT7+RBS+GFP+TT


Step 1 We minipreped the successful ligation of the pcstA (BBa_K118011) +RBS-T7RNAP (BBa_I712069) ligation which is our BioBrick BBa_K729007

Method

Miniprep Protocol 1 (Qiagen)
Step 1 - Pellet Cells: Pellet 1-5ml bacterial overnight culture by centrifugation at >8000rpm (68800 x g) for 3 min at room temperature (15-25oC)

Step 2 - Resuspend Cells: Resuspend pelleted bacterial cells in 250ul Buffer P1 and transfer to a microcentrifuge tube

Step 3 - Puncturing Cell Membrane: Add 250ul Buffer P2 and mix thoroughly by inverting the tube 4-6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 min.

Step 4 - Neutralising buffer P2: Add 350ul Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.

Step 5 - Centrifuge:

RPM: 13000

Time:10 minutes

Temperature: 18oC

Step 6 - Centrifuge: Apply the supernatant from step 5 to the QIAprep spin column by decanting or pipetting. Centrifuge for 30-60s and discard the flow-through.

Step 7 - Remove Endonucleases from Sample: Wash the QIAprep spin column by adding 500ul of Buffer PB. Centrifuge for 30-60s and discard flow-through.

Step 8 - Remove salts from sample: Wash the QIAprep spin column by adding 750ul of Buffer PE. Centrifuge for 3-60s and discard flow through.

Step 9 - Centrifuge:

RPM: 13000

Time:1 minute

Temperature: 18oC

Step 10 - Elute DNA: Place the QIAprep column in a clean 1.5ml microcentrifuge tube. To elute DNA, add 50ul Buffer EB to the centre of the spin column, let it stand for 1 min, and centrifuge for 1 min.


Step 2 Ligation was performed using the BioBricks BBa_I746909 and BBa_K729007 to generate a BioBrick that contains pcstA-T7RNAP-pT7-GFP-TT.


Step 3 Transformation – we plated 4 plates; plate 1 and 2 were with the transformed BBa_I746909 biobrick and plate 3 and 4 were transformed with our ligation (BBa_I746909+BBa_K729007). We are going to incubate over night at 37C and colonies picking the next day.

Method:

Transformation Protocol 1

Step 1 - Addition of BioBrick: To the still frozen competent cells, add 1 - 5 µL of the resuspended DNA to the 2ml tube.

Step 4 - Incubation: Close tube and incubate the cells on ice for 45 minutes.

Step 5 - Heat Shock: Heat shock the cells by immersion in a pre-heated water bath at 37ºC for 10 minutes.

Step 6 - Incubation: Incubate the cells on ice for 2 minutes.

Step 7 - Add media: Add 1.5ml of Lysogeny Broth and transfer to a falcon tube.

Step 8 - Incubation: Incubate the cells at 37ºC for 1 hour at RPM 550. 

Step 9 - Transfer: transfer the solution back into a 1.5ml Eppendorf and centrifuge

RPM: 14000

Time: 2 minutes

Temperature (18-25oC)

Step 10 - Resuspend:Remove supernatant and resuspend in 100ul LB

Step 11 - Plating: Spread the resuspended cell solution onto a selective nutrient agar plate. Place the plates in a 37°C static incubator, leave overnight (alternatively a 30°C static incubator over the weekend)

Wednesday (19.09.12)

Aim 1 – Check the result of transformation. We expect to see growth on plates with transformed biobricks and ligations.


Results - The table below indicates that there was growth on all of the plates


Plate Samples Volume Inoculated Colony Formation
1 BioBrick BBa_I746909 10ul Yes
2 BioBrick BBa_I746909 90uL Yes
3 Ligation BBa_I746909+BBa_K729007 10ul Yes
4 Ligation BBa_I746909+BBa_K729007 90uL Yes


Traformationweek15.png


Conclusion : The transformations have been successful. Colonies will be picked and inoculated into LB media for overnight culture


Step 4 – Inoculating Colonies into a Selective Broth. We had colonies on all the plates. We picked we picked 2 colonies from plates 1 and 2 and 4 colonies from plate 3 and 4. We grow all 12 colonies overnight.The table below indicates the volume of broth and the concentration of antibiotic required.

Picking Colonies
Step 1 – Creating culture media: In a sterile environment, set up X numbers of falcons, each with 5mls of media.


Step 2 - Inoculating Colonies into a Selective Broth:: Add Yul of antibiotic to reach desired antibiotic concentration.


(For Ampicillin this is 50ug/ml, For Kanamycin it is 25ug/ml, for Tetracycline it is 15ug/ml, and for Chloramphenicol it is 25ug/ml)


Step 4 – Selecting a Colony: Select a clear, isolated colony and using an inoculation hoop scoop up a colony onto the tip. Deposit in the falcon tube


Step 5 - Culture: Culture your falcon tubes overnight at a temperature of 37 oC. Leave for no longer than 16 hours.


Falcon tube Samples Broth Antibiotic (50 nu/uL)
1 BioBrick BBa_I746909 Lysogeny Broth Ampicillinn
2 BioBrick BBa_I746909 Lysogeny Broth Ampicillin
3 BioBrick BBa_I746909 Lysogeny Broth Ampicillin
4 BioBrick BBa_I746909 Lysogeny Broth Ampicillin
5 Ligation BBa_I746909+BBa_K729007 Lysogeny Broth Chloramphenicol
6 Ligation BBa_I746909+BBa_K729007 Lysogeny Broth Chloramphenicol
7 Ligation BBa_I746909+BBa_K729007 Lysogeny Broth Chloramphenicol
8 Ligation BBa_I746909+BBa_K729007 Lysogeny Broth Chloramphenicol
9 Ligation BBa_I746909+BBa_K729007 Lysogeny Broth Chloramphenicol
10Ligation BBa_I746909+BBa_K729007 Lysogeny Broth Chloramphenicol
11 Ligation BBa_I746909+BBa_K729007 Lysogeny Broth Chloramphenicol
12 Ligation BBa_I746909+BBa_K729007 Lysogeny BrothChloramphenicol

Thursday (20.09.12)

Aim - We want to check if the ligations were successful.


All 12 colonies so we are going to miniprep and nanodrop all of them

Step 1 - Miniprep

Method

Miniprep Protocol 1 (Qiagen)
Step 1 - Pellet Cells: Pellet 1-5ml bacterial overnight culture by centrifugation at >8000rpm (68800 x g) for 3 min at room temperature (15-25oC)

Step 2 - Resuspend Cells: Resuspend pelleted bacterial cells in 250ul Buffer P1 and transfer to a microcentrifuge tube

Step 3 - Puncturing Cell Membrane: Add 250ul Buffer P2 and mix thoroughly by inverting the tube 4-6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 min.

Step 4 - Neutralising buffer P2: Add 350ul Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.

Step 5 - Centrifuge:

RPM: 13000

Time:10 minutes

Temperature: 18oC

Step 6 - Centrifuge: Apply the supernatant from step 5 to the QIAprep spin column by decanting or pipetting. Centrifuge for 30-60s and discard the flow-through.

Step 7 - Remove Endonucleases from Sample: Wash the QIAprep spin column by adding 500ul of Buffer PB. Centrifuge for 30-60s and discard flow-through.

Step 8 - Remove salts from sample: Wash the QIAprep spin column by adding 750ul of Buffer PE. Centrifuge for 3-60s and discard flow through.

Step 9 - Centrifuge:

RPM: 13000

Time:1 minute

Temperature: 18oC

Step 10 - Elute DNA: Place the QIAprep column in a clean 1.5ml microcentrifuge tube. To elute DNA, add 50ul Buffer EB to the centre of the spin column, let it stand for 1 min, and centrifuge for 1 min.

Step 2 - Nanodrop

Method

Nanodrop Protocol

Software ND-1000 Model:

Step 1: Initialise the spectrophotometer by pipetting 1 µ of clean water onto lower optic surface, lowering the lever arm and selecting ‘initialise’ in the ND-1000 software

Step 2: Wipe and add elution buffer as negative control. Click blank in ND-1000 software

Step 3: Wipe and add 1 µl sample

Step 4: On the software set lambda to 260nm

Step 5: Lower the lever arm and click measure in ND-1000 software

Step 6: Take readings for concentration and purity

Step 7: Once measurement complete, wipe surface


In the table below are summarised all the concentrations obtained from the nanodrop.


Falcon tube Samples Plate Concentration at λ260 (ng/uL)
1 BioBrick BBa_I746909 1 188
2 BioBrick BBa_I746909 1 276.2
3 BioBrick BBa_I746909 2 194.6
4 BioBrick BBa_I746909 2 329.6
5 Ligation BBa_I746909+BBa_K729007 3 233.6
6 Ligation BBa_I746909+BBa_K729007 3 197.7
7 Ligation BBa_I746909+BBa_K729007 3 438.5
8 Ligation BBa_I746909+BBa_K729007 3 337.8
9 Ligation BBa_I746909+BBa_K729007 4 310.3
10Ligation BBa_I746909+BBa_K729007 4 244.3
11 Ligation BBa_I746909+BBa_K729007 4 249.8
12 Ligation BBa_I746909+BBa_K729007 4340.2


Step 3 - We carried out analytical digest of the minipreped DNA

Methods

Restriction Enzyme Digest Protocol 1

Step 1 - Thawing cells: Thaw all materials on ice

Step 2 - Adding Ingredient: Add the following ingredients to autoclaved/sterile eppendorf tubes

Component Amount (ul) (one enzyme used) Amount (ul) (two enzymes used)
dH20 2.5 1.5
Buffer 1x 1 1
DNA template 5 5
BSA 0.5 0.5
Enzyme 1 1 2
Enzyme 2 N/A 1


Step 3 - Addition of BioBrick: Flick contents gently and centrifuge.

Step 4 - Centrifuge:

RPM: 14000

Time: 1 minute

Temperature: 18oC

Step 5 - Digest Program: Place the samples on a thermocycler under the following conditions:

RPM: 550

Time: 2.5 hours

Temperature: 37oC

Step 6 - Denaturing Enzymes: If you are not running the samples on a gel immediately, denature the restriction enzymes by running the samples on a thermocycler under the following conditions:

RPM: 550

Time: 25 minutes

Temperature: 65oC


step 4 - Gel electrophoresis of the analytical digest to see if we have the right DNA size


Gel3table1week15.png


L - DNA ladder 10037 bp (5μL)

1 - PstI cut BBa_I746909+PSB1A3

2 - PstI and Xbal cut BBa_I746909+PSB1A3

3 - PstI cut BBa_I746909+PSB1A3

4 - PstI and Xbal cut BBa_I746909+PSB1A3

5 - PstI cut BBa_I746909+PSB1A3

6 - PstI and Xbal cut BBa_I746909+PSB1A3

7 - PstI cut BBa_I746909+PSB1A3

8 - PstI and Xbal cut BBa_I746909+PSB1A3

9 – PstI cut ligation (BBa_K729007+BBa_I746909) + PSB1C3

10 - PstI and Xbal cut ligation (BBa_K729007+BBa_I746909) + PSB1C3

11 - PstI cut ligation (BBa_K729007+BBa_I746909) + PSB1C3

12 - PstI and Xbal cut ligation (BBa_K729007+BBa_I746909) + PSB1C3

L DNA ladder 10037 bp (5μL)

13 - PstI cut ligation (BBa_K729007+BBa_I746909) + PSB1C3

14 - PstI and Xbal cut ligation (BBa_K729007+BBa_I746909) + PSB1C3

15 - PstI cut ligation (BBa_K729007+BBa_I746909) + PSB1C3

16 - PstI and Xbal cut ligation (BBa_K729007+BBa_I746909) + PSB1C3

17 - PstI cut ligation (BBa_K729007+BBa_I746909) + PSB1C3

18 - PstI and Xbal cut ligation (BBa_K729007+BBa_I746909) + PSB1C3


Gel3table2week15.png


L - DNA ladder 10037 bp (5μL)

19 - PstI cut ligation (BBa_K729007+BBa_I746909) + pSB1C3

20 - PstI cut cut ligation (BBa_K729007+BBa_I746909) + pSB1C3

21 - PstI and Xbal cut ligation (BBa_K729007+BBa_I746909) + pSB1C3

22 - PstI cut ligation (BBa_K729007+BBa_I746909) + pSB1C3

23 -PstI and Xbal cut ligation (BBa_K729007+BBa_I746909) + pSB1C3


BioBricks BioBrick size (bp) Ligation size (bp) Plasmid backbonePasmid backbone(bp)Combined size(bp)
BBa_K729007+BBa_I746909 2817+906 3731 pSB1C320705801
BBa_I746909 906 pSB1A3 2155 3061


Conclusion - The ligation was successful the bands on the gel correspond to the bands we were expecting.

15-5

Tuesday (18.09.12)

Aim – We would like to determine if BBa_K729006 is functioning as intended and producing laccase.

Method:

Laccase characterisation
In order to characterise laccase, the following steps are carried out:


1) W3110 cells transformed with laccase and control W3110 cells are inoculated in 10mL of LB media, and incubated overnight at 37˚C in a 200rpm shaker


2) The O/N culture is then centrifuged at 6100g for 20 minutes, in order to extract the media containing laccase. The supernatant is retained.


3) In order to take readings, cuvettes are prepared with 2.2mL KH2PO4 buffer + 0.5mL Laccase supernatant + 0.3mL Syringaldazine, which is added immediately before readings are begun (as explained in step 4). A blank is created using 0.5mL of LB media instead of laccase supernatant.


4) The optical density at 530nm of each of the samples is measured at five minute intervals over 30 minutes.


Conclusion - Our cells are producing an elevate amount of laccase compared to the control, indicating that the BioBrick is functioning as intended.

Friday (21.09.12)

Aim – We would like to determine if our laccase producing BBa_K729006 is capable of degrading polyethylene.

1) Cells are grown overnight in 10ml of LB media at 37˚C, 200rpm.

a) BBa_K729006
b) W3110 Control

2) 1cm2 low density polyethylene squares are introduced into the media, which is incubated for 3 days. Separate LDPE squares are placed in water in this timeframe.

3) LDPE squares are removed from media, dried, mounted, and coated with gold-palladium for scanning electron microscopy

4) LDPE samples are viewed under the SEM at 100x and 10 000x magnification, observing pitting caused by laccase catalysed degradation.


Conclusion - Our laccase producing cells are degrading polyethylene, allowing our BioBrick to be utilised for this function.


Sunday (14.09.12)

Aim – We would like to determine if BBa_K729005 is functioning as intended.

1) Cells are grown overnight in 15ml of LB media at 37˚C, 200rpm.

a) BBa_K729005
b) W3110 Control

2) Shake flasks are inoculated with 5 ml of cells, with the following media (50ml):

a) LB media
b) LB media + 0.3M NaCl
c) LB media + 0.6M NaCl

3) Shake flasks are run at 37˚C, 200rpm, measuring the optical density at 600 nm for each flask each hour.

4) Optical density is measured until the cells exit the exponential growth phase


Conclusion - Our transformed cells exhibit improved growth rates, and higher cell count in high salt conditions. This indicates that our cells are producing IrrE, and that our BioBrick is functioning as intended.