Team:University College London/LabBook/Week8

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Contents

Monday 30.6.12

Aim: Repeat the gel from Expt 7.3 (week 7) with a smaller ladder and higher agarose percentage in order to detect the very small inserts of BBa_J23119 (35bp) and BBa_B0034 (12hp). Previous ladder used was 10037 bp, and this time we used a 25bp ladder.

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 - Setting up Digests and Controls: The protocol describes the recipe for (i) Digested Plasmid and (ii) Uncut Control. The table below indicates that an uncut and an Xba1/Spe1 digested sample be set up for each BioBrick. Set up Eppendorfs as follows


Samples Recipe Enzymes
BioBrick BBa_J23119 Digested Plasmid Xba1 & Spe1
Undigested Plasmid (Control) None
BBa_B0034 Digested Plasmid Xba1 & Spe1
Undigested Plasmid (Control) None


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:The image below shows a 3.5% Agarose Gel of an Analytical Restriction Enzyme Digest for Expt 7.3. The high agarose concentration of the gel was intended to slow the progression of DNA fragments to enable us to detect the small inserts of BBa_J23119 and BBa_B0034. The table below indicates the expected sizes of BioBricks and Plasmid Backbones. Accordingly, we used a the smallest available ladder, with fragments ranging from 25bp to 500bp. At the top of the gel it is possible to see the large fragments we detected on a 1000bp gel in Expt 7.3 Week 7. However, we had expected this gel to also demonstrate the BBa_J23119 insert (35bp) corresponding to letter A in Lane 1, and the BBa_B0034 insert, corresponding to letter B in Lane 3. No product was found. As expected, there was no product measurable against the 25bp ladder for the uncut plasmids in Lane 2 and Lane 4.


BioBrick Expected BioBrick Size (bp) Plasmid Backbone Plasmid Expected Size (bp) Combined Size
BBa_J23119 35 PSB1A2 2079 2114
BBa_B0034 12 PSB1A2 2079 2091


Conclusion:No product was detected for either BioBrick insert. However, we do no feel this necessarily proves the transformation failed, especially as we detected the correct plasmid backbone for each in week 7 (Expt 7.3). The modest nanodrop concentration obtained last week suggests that the concentration was not high enough to allow such a small fragment to be visualised. We are considering alternatives such as running a polyacrylamide gel, which is more sensitive to small fragments, or amplifying the fragment using PCR

Monday 30.7.12

Aim - Picking colonies: Colonies were picked for BBa_I750016, which demonstrated colony formation in Expt 7.4 on Friday 27.7.12.

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.

Step 2 – Inoculating Colonies into a Selective Broth: The table below indicates the volume of broth and the concentration of antibiotic required for each BioBrick.

Samples Volume Inoculated Broth (ml) Antibiotic (ug/ml)
BioBrick BBa_I750016 10ul Lysogeny Broth (5) Ampicillin(50ug/ml)
90ul


Tuesday 31.7.12

Aim – Results from Colony Picking

Results: The table below indicates whether there was growth for BBa_I1750016


Samples Volume Inoculated Colony Formation
BioBrick BBa_I1750016 10μl Yes
90μl Yes

Conclusion: We can proceed onto Miniprep, Analytical Digest, and Nanodrop.

Method

Miniprep:

Miniprep Protocol 1 (ANACHEM)

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


Wednesday 1.8.12

Aim: Nanodrop of BBa_I1750016

BioBrick λ260 λ 280
BBa_I750016 (ng/μl) 7.6 7.6

Conclusion: The concentration of BioBrick is to low to justify running a gel. We conclude the transformation failed.

Monday 30.7.12

Aim - Transformation of previously failed BioBricks: BBa_C0040 was transformed in Expt 7.4, but appeared to be contaminated. BBa_R0040, as part of the same experiment, did not form colonies at all after transformation. Therefore both were reattempted.


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)


Step 1 – Thawing Cells: Use W3100 cell line created in Week 2 (Expt 2.1)

Step 3 – Addition of BioBrick: To each 2ml eppendorf, add 1ul of the following BioBricks. Include an extra tube as a control, with no BioBrick added

Function Module
BioBrick BBa_C0040 Tetracycline Repressor Buoyancy
BBa_R0040 TetR Repressible Promoter Buoyancy
Control Positive (one for each of the above BioBricks)
Negative (No BioBrick)

Step 9 – Plating samples on Agar Plates: The table below indicates the chosen inoculation volume (two for each BioBrick) and the correct gel antibiotic concentration for all samples.(Extra caution was taken to allow agar to cool before adding Ampicillin, in case this is the cause of difficulty).

Samples Volume Inoculated Antibiotic in Gel (ug/ml)
BioBrick BBa_C0040 10ul Ampicillin(50ug/ml)
90ul
BBa_R0040 10ul
90ul
Control Positive (Contains BioBrick BBa_C0040) 36ul No Antibiotic
Negative (No BioBrick) 36ul 1x Ampicillin(50ug/ml)

Tuesday 31.8.12

Aim - Check results of Transformation: The table below indicates whether there was growth on the Agar Plates after Transformation.


Samples Volume Inoculated Colony Formation
BioBrick BBa_C0040 10ul Yes
90ul Yes
BioBrick BBa_R0040 10ul Yes
90ul Yes
Control Positive (Contains BioBrick BBa_C0040) 36ul Yes
Negative (No BioBrick) 36ul No


Conclusion: Transformation was successful so will proceed to Colony Picking

Method

Aim - Picking colonies:

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.

Step 2 – Inoculating Colonies into a Selective Broth: The table below indicates the volume of broth and the concentration of antibiotic required for each BioBrick.

Samples Volume Inoculated Broth (ml) Antibiotic (ug/ml)
BioBrick BBa_C0040 10ul Lysogeny Broth (5) Ampicillin(50ug/ml)
90ul
BBa_R0040 10ul
90ul


Wednesday 1.8.12

Aim – Results from Colony Picking

Results: The table below indicates whether there was growth for the BioBricks


Samples Volume Inoculated Growth
BioBrick BBa_C0040 10μl Yes
90μl Yes
BBa_R0040 10μl Yes
90μl Yes


Conclusion: We can move on to miniprep

Methods

Miniprep of Samples

Miniprep Protocol 1 (ANACHEM)

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

Nanodrop

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

Results:

BioBrick λ260 λ 280
BBa_C0040 (ng/μl) 9.4 18.9
BBa_R0040 (ng/μl) 9.3 36.9

Conclusion: The concentration of these plasmids is very low. We can conclude that the transformation has failed. The reasons for the continued failure of these plasmids are unclear

Tuesday 31.7.12

Aim: to generate enough plasmid backbones (PSB1K3, PSB1C3, PSB1A3) that we could use for 3A ligation.

PCR Protocol


Tuesday 31.7.12

Aim: In order to set up our salt tolerance characterisation we must grow up 10ml of W3110 and Salt Tolerance (BBa_K398108) transformed W3110 overnight


Wednesday 1.8.12

Aim: Today we commence the protocols for characterising salt tolerance.

Results: Our results demonstrate that out transformed cells are salt tolerant, compared to the untransformed W3100 cells.Our results reflect those of TU Delft 2010 who characterised the same BioBrick. However they did not show their results beyond the exponential phase, which we have included.



Conclusions: Growth rate during exponential phase is greater in the K398108 transformed E.coli W3110 than in the untransformed (same conclusion as drawn by TU Delft 2010). Increase in growth rates significantly lower than original at highest salt concentration – could be explained by use of different E.coli strains i.e. higher growth rate of the non-transformed strain. We plan to repeat to strengthen conclusions.


Wednesday 1.8.12

Aim: Start 3A assembly to ligate the first (BBa_J23119+BBa_B0034), and second (plasmid backbone PSB1K3 and Pcst starvation promoter) constructs.

Protocols?

Thursday 2.8.12

Aim: Evaluation of the plates from 3A ligation. This should inform us as to whether we can proceed and do the colony picking of both A (J23119+BB0034), B (Pcst + BB0034) constructs.

Result: Observations showed that 3A was successful for the construct A. However there was no growth assembled J23119+BB0034, but there was no growth for the construct B. The following table summarises the results.

Conclusion: Colony picking was carried out for the first construct (BBa_J23119+BBa_B0034), but not from the second construct (plasmid backbone PSB1K3 and Pcst starvation promoter).

8-5