Team:University College London/LabBook/Week13

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'''Methods:'''
'''Methods:'''
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For Wnu cell line which has native secreted nuclease activity
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1. Prepare 11-16 plates (10ml LBAgar+10ul AMP +10 ul 1M IPTG). IPTG induces the lac promoter which in turn activates the transcription of nuclease.
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2. Streak cells onto all plates at the same time
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3. Incubate at 37°C
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4. Apply hydrochloric acid (HCL) to the first plate before putting them in the incubator (set time as zero)
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5. Take a second reading after four hours, followed by six readings every 3 hours, and a final three readings every two hours.
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6. When the reading is taken, observe the following:
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a) Diameter of the colony (once the diameter of the colony is measured, pick the colony and put it to grow in LB for nine hours)
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b) Diameter of the halo that is achieved once HCL is applied
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c) OD from a)
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d) Estimate of the depth of the colony on the agar plate
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For BL21 cell line that has been modified to contain nuclease
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7. Prepare 11-16 plates (LB Agar + CMP)
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8. Streak cells onto all plates at the same time
 +
9. Incubate at 37°C
 +
10. Apply HCL to the first plate before putting in the incubator (set time as zero)
 +
11. Take a second reading after four hours, followed by six readings every 3 hours, and a final  three readings every two hours.
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12. When the reading is taken, observe the following:
 +
a. Diameter of the colony (once the diameter of the colony is measured, pick the colony and put it to grow in LB for nine hours)
 +
b. Diameter of the halo that is achieved once HCL is applied
 +
c. OD from a)
 +
d. Estimate of the depth of the colony on the agar plate
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In order to be able to characterise cells, the following protocol was used:
 
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<html><div class="protocol protocol-Generic">Nuclease characterisation</div><div class="protocolContent"></html>{{:Team:University_College_London/Week11YanikaA}}<html></div></html>
 

Revision as of 03:18, 27 September 2012

Contents

Monday 03/09

Aim:


To repeat the DNAse/Nuclease Halo experiment in order to check whether results are repeatable. This included collecting data of halo diameters and colony diameters over 28 hour, and to determine how the nuclease production increases overtime.


Methods:

For Wnu cell line which has native secreted nuclease activity 1. Prepare 11-16 plates (10ml LBAgar+10ul AMP +10 ul 1M IPTG). IPTG induces the lac promoter which in turn activates the transcription of nuclease. 2. Streak cells onto all plates at the same time 3. Incubate at 37°C 4. Apply hydrochloric acid (HCL) to the first plate before putting them in the incubator (set time as zero) 5. Take a second reading after four hours, followed by six readings every 3 hours, and a final three readings every two hours. 6. When the reading is taken, observe the following: a) Diameter of the colony (once the diameter of the colony is measured, pick the colony and put it to grow in LB for nine hours) b) Diameter of the halo that is achieved once HCL is applied c) OD from a) d) Estimate of the depth of the colony on the agar plate For BL21 cell line that has been modified to contain nuclease 7. Prepare 11-16 plates (LB Agar + CMP) 8. Streak cells onto all plates at the same time 9. Incubate at 37°C 10. Apply HCL to the first plate before putting in the incubator (set time as zero) 11. Take a second reading after four hours, followed by six readings every 3 hours, and a final three readings every two hours. 12. When the reading is taken, observe the following: a. Diameter of the colony (once the diameter of the colony is measured, pick the colony and put it to grow in LB for nine hours) b. Diameter of the halo that is achieved once HCL is applied c. OD from a) d. Estimate of the depth of the colony on the agar plate


Results:


The following table images show the results of the DNase assay:


2 3 4 5 6 7 8 9 10 11 The following table shows the colony and halo diameters, plus the OD of one plate at different sampling times. Date Time Colony diameter Halo diameter Absorbance at OD600 03.09.2012 12.30pm 0 0 0 03.09.2012 16.30pm 0 0 0 03.09.2012 19.30 pm 0 0 0 03.09.2012 22.30 pm 0.5mm 1mm 0.002 04.09.2012 01.30 am 1mm 3.5mm 0.182 04.09.2012 04.30 am 1.5mm 7mm 0.238 04.09.2012 07.30 am 2mm 9mm 0.297 04.09.2012 10.30am 2.5mm 11mm 0.518 04.09.2012 12.30pm 3mm 12.5mm 0.701 04.09.2012 14.30pm 3.5mm 14mm 0.811 04.09.2012 16.30pm 4mm 15mm 0.906 Conclusion: We have obtained data consistent with the previous DNase assay conducted, and thereby conclude that a linear correlation exists between colony size and halo diameter.

Tuesday 04/09

Aim:


To pick colonies from all the ligation transformation plates, prepared in Week 12. This included the ligations of curli, laccase and nuclease to the linker and the pb. This was done in order to purify the DNA and use it in subsequent ligations. This was done because gels from Week 12 showed unexpected bands. Hence, it was necessary to check whether this was due to unsuccessful purification and analytical digest, or due to unsuccessful ligation.


Methods:


Colony Picking
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.

Wednesday 05/09

Step 1 - Aim:


To carry out a miniprep of the inoculations from the ligation transformation plates. After this, an analytical digest is prepared and run on a gel in order to confirm DNA identity


Method:


In order to carry out a miniprep, the following protocol was used:


MiniPrep

Step 1 - Pellet Cells: Pellet 1.5-5ml bacterial culture containing the plasmid by centrifugation g = 6000

Time = 2 min

Temperature = (15-25oC)

Step 2 - Resuspend Cells: Add 250ul S1 to the pellet and resuspend the cells completely by vortexing or pipetting. Transfer the suspension to a clean 1.5ml microcentrifuge tube.

Step 3 - Puncturing Cell Membrane: Add 250ul S2 gently mix by inverting the tube 4-6 times to obtain a clear lysate. Incubate on ice or at room temperature for NOT longer than 5 min.

Step 4 - Neutralising S2: Add 400ul Buffer NB and gently mix by inverting the tube 6-10 times, until a white precipitate forms.

Step 5 - Centrifuge:

g: 14000

Time:10 minutes

Temperature: (15-25oC)

Step 6 - Centrifuge: Transfer the supernatant into a column assembled in a clean collection tube (provided. Centrifuge:

g = 10,000

Time: 1 minute

Temperature: (15-25oC)

Step 7 - Wash Column: Discard the flow-through and wash the spin column by adding 700ul of Wash Buffer. Centrifuge:

g - 10,000

Time - 1 minute

Temperature: (15-25oC)

Step 8 - Remove residual ethanol: Centrifuge:

g - 10,000

Time - 1 minute

Temperature: (15-25oC)

Step 9 - Elute DNA: Place the column into a clean microcentrifuge tube. Add 50-100ul of Elution Buffer, TE buffer or sterile water directly onto column membrane and stand for 1 min. Centrifuge:

g - 10,000

Time - 1 minute

Temperature: (15-25oC)

Step 10 - Storage: Store DNA at 4oC or -20oC


In order to carry out an analytical digest, the following protocol was used:


Analytical Digest
Step 1 - Thawing cells: Thaw all materials on ice

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

Insert Table

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: 1 hour

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

Temperature: 18oC


In order to run digest samples on a gel, the following protocol was used:


Gel electrophoresis

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 not shown


Conclusion:


Since band sizes were as expected, the DNA was taken forward and used in a preparative digest in preparation of ligation.


Step 2 - Aim:


To carry out a preparative digest on the purified DNA (Where did we day that we carried out PCR???)


Methods:


The following table shows the DNA part and the enzymes used to digest it.

No Part Enzymes
1 laccase X+P
2 curli X+P
3 irrE X+P
4 irrE E+P
5 nuclease X+P
6 nuclease X+P
7 plasmid backbone E+P
8 Plasmid backbone E+P
9 Linker E+S


Step 3 - Aim:


To carry out ligations using the inserts and backbone prepared previously. The ligations carried out included qoui???? Vat iz zis writing down here?

X+P cut to E+P cut Plasmid All.

X+S Plasmid backbone religated control and no ligase control.

E+P cut ire to E+P cut plasmid backbone.


Methods:


Quick T4 Ligase was used to carry out ligations. The protocol followed in order to carry out ligations was as supplied with the reagants. Do we need to write it down?


Step 4 - Aim:


To carry out a transformation of the ligation


Methods:


Protocol? 5uL used to transform 100uL cells.

Thursday 06/09

Aim:


To check whether transformations using ligation product on the previous day were successful.


Results:


No colonies were visible on the plates. Hence, the transformation was considered to be unsuccessful and needs to be repeated.



Wednesday 05/09

Aim:

To characterise the two strains of laccase obtained from the laccase ligation.


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.


Result:


Not shown


Conclusion:


We have determined that laccase strain 2 is a better laccase producing strain. Furthermore, we have proven the function of our BioBrick, as the oxidation activity of both laccase stratins was significantly higher than that of the control W3110 strain.


Thursday 06/09

Aim:


To compare the difference in growth of Indoli and E. coli growth in LB & MB media


Methods:


Growth rate characterisation
In order to compare growth rates of W3110 and indolifex in LB and MB media, the following steps were carried out:


1. Inoculate 10ul of W3110 glycerol stock in 10ml of LB and another 10ul of W3110 glycerol stock in 10ml of MB


2. Repeat this with an indolifex glycerol stock


3. Measure the absorbence of all four samples every hour for 12 hours. Then take a final measurement after another 12 hours. Thus, measurements span 24 hours.


Results:


The following graphs show growth of the different strains and different media. The flasks were placed in a 370C shaker at 200rpm.


UCL2012GROWTHCOMPARE.png


Conclusion:


From the above graphs, it can be seen that marine bacteria grows better in MB, irrespective of the temperature used.


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