Team:University College London/LabBook/Week8

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Latest revision as of 01:23, 27 September 2012

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
	BBa_J23119	Undigested Plasmid (Control)	None
	BBa_B0034	Digested Plasmid	Xba1 & Spe1
	BBa_B0034	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)
	BBa_I750016	90ul

Tuesday 31.7.12

Aim – Results from Colony Picking

Results: The table below indicates whether there was growth for BBa_I750016

Samples		Volume Inoculated	Colony Formation
BioBrick	BBa_I750016	10μl	Yes
	BBa_I750016	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_I750016

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 W3110 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
BioBrick	BBa_R0040	TetR Repressible Promoter	Buoyancy
Control	Positive (one for each of the above BioBricks)
Control	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)
	BBa_C0040	90ul
	BBa_R0040	10ul
	BBa_R0040	90ul
Control	Positive (Contains BioBrick BBa_C0040)	36ul	No Antibiotic
Control	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
BioBrick	BBa_C0040	90ul	Yes
BioBrick	BBa_R0040	10ul	Yes
BioBrick	BBa_R0040	90ul	Yes
Control	Positive (Contains BioBrick BBa_C0040)	36ul	Yes
Control	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)
	BBa_C0040	90ul
	BBa_R0040	10ul
	BBa_R0040	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
	BBa_C0040	90μl	Yes
	BBa_R0040	10μl	Yes
	BBa_R0040	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. After the PCR reaction, we can detect the presence of the correct products by running a 1% gel electrophoresis.

Method:

PCR Protocol

Step 1 - Setting up PCR tubes: Thaw reagents and add to PCR tubes in the proportions described in the table below

PCR Components	Volume (ul)
5x Reaction Buffer	10
25mM MgCl2	4
10mM dNTPs	1
10uM Forward primer	5
10uM Reverse primer	5
DNA Polymerase	0.25
Nuclease Free Water	24.25
Template DNA	0.5
Total Volume	0.5

Step 2 - PCR program: Add PCR tubes to a thermocycler and run under the following conditions.

PCR conditions	Temp (^oC)	Time (s)
Initial Denaturation (1 cycle)	95	30
Denaturation/Annealing/Extension (30 cycles)	95/55/72	10/25/120
Final Extension (1 cycle)	72	600
Hold	4	∞

Wednesday 1.8.12

Aim: to run a 1% gel of the products of yesterdays PCR reaction.

Method

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 1% Agarose Gel of an Analytical Restriction Enzyme Digest for Expt 8.2. Lane 1, 2 and 3 all show products of approximately 2000bp which is of the expected size for the plasmid backbones PSB1K3 (indicated by A), PSB1C3 (indicated by B) and PSB1A3 (indicated by C).

Plasmid Backbone	Expected Size bp
PSB1K3 (Kanamycin)	2000
PSB1C3 (Chloramphenicol)	2000
PSB1A3 (Ampicillin)	2000

Conclusion:The PCR was successful.

Friday 3.8.12

Aim: PCR clean up for the plasmid backbones and nanodrop.

Methods:

PCR clean up

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: Nanodrop concentrations are presented in the table below. All are low except for the backbone containing kanamycin. We concluded that PCR for these backbones needs to be repeated

BioBrick	λ260	λ 280
PSB1A3 (ng/μl)	27.3	26.6
PSB1C3 (ng/μl)	23	24.4
PSB1K3 (ng/μl)	66.5	87

Conclusion: The PCR for PSB1A3 and PSB1C3 need to be repeated.

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 W3110 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.

Method

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) and B (Pcst + BB0034) constructs.

Result: Observations showed that 3A was successful for the construct A, as shown in the image below. However there was no growth assembled pCST+BBa_B0034 (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).

Method

Picking Colonies Protocol

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.

Friday 3.8.12

Aim:Check the results of colony picking.

Results: There was growth. Next we can proceed to run a gel and a nanodrop to detect the presence of the correct bands, and whether we have a useable concentration.

Thursday 2.8.12

Aim - Transformation of alternative BioBricks: We have colony picked the BBa_J23119 constitutive promoter and the BBa_B0034 Ribosome Binding Site twice (Expt 6.3 & Expt 7,3), and gained a reasonable plasmid concentration. However the concentration was not high enough to be able to detect the product on the gel. This concerns us, because although we have detected the correct plasmid backbone, we are worried that we lack evidence that the transformation was successful. One option is to attempt several other BioBricks with the same function, in order to increase the chances of generating a product on the gel - and confirming that we definitely have a BioBricks with the required function.It would also increase the chance of generating successful ligations if we can reach a higher concentration of plasmid. We will also be reattempting the transformation of BBa_I750016 which failed several attempts at colony picking.

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 W3110 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_J23100	Constitutive Promoter	All
	BBa_J23106	Constitutive Promoter	All
	BBa_B0030	Ribosome Binding Site	All
	BBa_I750016	Ribosome Binding Site	All
Control	Positive (one for each of the above BioBricks)
Control	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_J23100	10ul	Ampicillin(50ug/ml)
	BBa_J23100	90ul
	BBa_J23106	10ul
	BBa_J23106	90ul
	BBa_B0030	10ul
	BBa_B0030	90ul
	BBa_I750016	10ul
	BBa_I750016	90ul
Control	Positive	36ul	No Antibiotic
Control	Negative (No BioBrick)	36ul	1x Ampicillin(50ug/ml)

Friday 3.8.12

Aim - Check results of Transformation: The table below indicates whether there was growth on the Agar Plates after Transformation. Included below are images of the Agar Plates for each BioBrick.

Samples		Volume Inoculated	Colony Formation
BioBrick	BBa_J23100	10ul	Yes
	BBa_J23100	90ul	Yes
	BBa_J23106	10ul	Yes
	BBa_J23106	90ul	Yes
	BBa_B0030	10ul	Yes
	BBa_B0030	90ul	Yes
	BBa_I750016	10ul	Yes
	BBa_I750016	90ul	Yes
Control	Positive (Contains BioBrick BBa_C0040)	36ul	Yes
Control	Negative (No BioBrick)	36ul	Yes

Conclusion: We had growth for all of our BioBricks, but also for the negative control which indicates there was contamination. Given we have had several issues with negative control recently, we decided we would not let the contamination delay the project. We will risk colony picking with the hope that we have some correctly transformed colonies. This will be commenced next week. It is worth noting that for Constitutive Promoter BBa_J23106 and for the Gas Vesicle Cluster BBa_I750016 there was a continuous film of growth. This suggests to us that the agar was not selective enough.

@@ Line 32: / Line 32: @@
 '''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.
-{{:Team:University_College_London/templates/pictureinsert|title=gel2|url=images/3/3f/Ucligem2012_gel8.1.png}}
+{{:Team:University_College_London/templates/pictureinsert|title=Picture 1|url=images/d/d5/Ucligem8.1.png}}
@@ Line 75: / Line 75: @@
 '''Aim – Results from Colony Picking'''
-'''Results:''' The table below indicates whether there was growth for BBa_I1750016
+'''Results:''' The table below indicates whether there was growth for BBa_I750016
@@ Line 82: / Line 82: @@
 ! colspan="2" |  Samples !! Volume Inoculated !! Colony Formation
 |-
-| rowspan="8" |BioBrick ||rowspan="2" | BBa_I1750016  || 10μl || Yes
+| rowspan="8" |BioBrick ||rowspan="2" | BBa_I750016  || 10μl || Yes
 |-
 | 90μl || Yes
+|}
 '''Conclusion:''' We can proceed onto Miniprep, Analytical Digest, and Nanodrop.
 '''Method'''
@@ Line 93: / Line 95: @@
 <html><div class="protocol protocol-Miniprep">Miniprep Protocol 1 (ANACHEM)</div><div class="protocolContent"></html>{{:Team:University_College_London/Protocols/Miniprep2}}<html></div></html>
-'''Restriction Digest:'''
-<html><div class="protocol protocol-RestrictionEnzymeDigest">Restriction Enzyme Digest Protocol 1</div><div class="protocolContent"></html>{{:Team:University_College_London/Protocols/RestrictionEnzymeDigest1}}<html></div></html>
-'''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
+== Wednesday 1.8.12 ==
+Aim: Nanodrop of BBa_I750016
 {| class="wikitable"
 |-
-! colspan="2" |  Samples  !!Recipe !! Enzymes
+! BioBrick !! λ260 !! λ 280
 |-
-| rowspan="7" |BioBrick || rowspan="2" | BBa_I150016 ||Digested Plasmid || Xba1 & Spe1
+| BBa_I750016 (ng/μl) || 7.6 || 7.6
 |-
-| Undigested Plasmid (Control) || None
 |}
+'''Conclusion:''' The concentration of BioBrick is to low to justify running a gel. We conclude the transformation failed.
 <html>
@@ Line 113: / Line 115: @@
 </html>
-== 8-1 ==
+== 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:'''
+<html><div class="protocol protocol-Transformation">Transformation Protocol 1</div><div class="protocolContent"></html>{{:Team:University_College_London/Protocols/Transformation1}}<html></div></html>
+'''Step 1 – Thawing Cells:''' Use W3110 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
+{| class="wikitable"
+|-
+!  colspan="2" |   !! Function !! Module
+|-
+| rowspan="2" |BioBrick|| BBa_C0040 || Tetracycline Repressor|| Buoyancy
+|-
+| BBa_R0040 || TetR Repressible Promoter || Buoyancy
+|-
+| rowspan="2"|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).
+{| class="wikitable"
+|-
+! colspan="2" |  Samples !! Volume Inoculated !! Antibiotic in Gel (ug/ml)
+|-
+| rowspan="4" |BioBrick ||rowspan="2" | BBa_C0040  || 10ul || rowspan="4" | Ampicillin(50ug/ml)
+|-
+| 90ul
+|-
+| rowspan="2" | BBa_R0040  || 10ul
+|-
+| 90ul
+|-
+| rowspan="2" | 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.
+{| class="wikitable"
+|-
+! colspan="2" |  Samples !! Volume Inoculated !! Colony Formation
+|-
+| rowspan="2" |BioBrick ||rowspan="2" | BBa_C0040  || 10ul || Yes
+|-
+| 90ul || Yes
+|-
+| rowspan="2" |BioBrick ||rowspan="2" | BBa_R0040  || 10ul || Yes
+|-
+| 90ul || Yes
+|-
+| rowspan="2" | 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:'''
+<html><div class="protocol protocol-ColPic">Picking Colonies</div><div class="protocolContent"></html>{{:Team:University_College_London/Protocols/ColPic}}<html></div></html>
+'''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.
+{| class="wikitable"
+|-
+! colspan="2" |  Samples !! Volume Inoculated !! Broth (ml) !! Antibiotic (ug/ml)
+|-
+| rowspan="8" |BioBrick ||rowspan="2" | BBa_C0040  || 10ul ||rowspan="8" |Lysogeny Broth (5) || rowspan="8" | Ampicillin(50ug/ml)
+|-
+| 90ul
+|-
+| rowspan="2" | 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
+{| class="wikitable"
+|-
+! colspan="2" |  Samples !! Volume Inoculated !! Growth
+|-
+| rowspan="8" |BioBrick ||rowspan="2" | BBa_C0040  || 10μl || Yes
+|-
+| 90μl || Yes
+|-
+| rowspan="2" | BBa_R0040  || 10μl || Yes
+|-
+| 90μl || Yes
+|}
+'''Conclusion:''' We can move on to miniprep
+'''Methods'''
+'''Miniprep of Samples'''
+<html><div class="protocol protocol-Miniprep">Miniprep Protocol 1 (ANACHEM)</div><div class="protocolContent"></html>{{:Team:University_College_London/Protocols/Miniprep2}}<html></div></html>
+'''Nanodrop'''
+<html><div class="protocol protocol-Nanodrop">Nanodrop Protocol</div><div class="protocolContent"></html>{{:Team:University_College_London/Protocols/Nanodrop}}<html></div></html>
+'''Results:'''
+{| class="wikitable"
+|-
+! 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
 <html>
 </div><div class="experiment"></div>
 <img id="8-2" src="https://static.igem.org/mediawiki/2012/a/a8/Ucl2012-labbook-graph8-2.png" /><div class="experimentContent">
 </html>
-== 8-2 ==
+== Tuesday 31.7.12 ==
+'''Aim:''' to generate enough plasmid backbones  (pSB1K3, pSB1C3, pSB1A3) that we could use for 3A ligation. After the PCR reaction, we can detect the presence of the correct products by running a 1% gel electrophoresis.
+'''Method:'''
+<html><div class="protocol protocol-PCR">PCR Protocol</div><div class="protocolContent"></html>{{:Team:University_College_London/Protocols/PCR}}<html></div></html>
+== Wednesday 1.8.12 ==
+'''Aim:''' to run a 1% gel of the products of yesterdays PCR reaction.
+'''Method'''
+<html><div class="protocol protocol-Electrophoresis">Electrophoresis Protocol</div><div class="protocolContent"></html>{{:Team:University_College_London/Protocols/Electrophoresis}}<html></div></html>
+'''Results:''' The image below shows a 1% Agarose Gel of an Analytical Restriction Enzyme Digest for Expt 8.2. Lane 1, 2 and 3 all show products of approximately 2000bp which is of the expected size for the plasmid backbones PSB1K3 (indicated by A), PSB1C3 (indicated by B) and PSB1A3 (indicated by C).
+{{:Team:University_College_London/templates/pictureinsert|title=Picture 1|url=images/e/e5/Ucligem20128.2.png}}
+{| class="wikitable"
+|-
+! Plasmid Backbone !! Expected Size bp
+|-
+| PSB1K3 (Kanamycin)  || 2000
+|-
+| PSB1C3 (Chloramphenicol)  || 2000
+|-
+| PSB1A3 (Ampicillin) || 2000
+|}
+'''Conclusion:'''The PCR was successful.'''
+== Friday 3.8.12 ==
+'''Aim:''' PCR clean up for the plasmid backbones and nanodrop.
+'''Methods:'''
+PCR clean up
+<html><div class="protocol protocol-Nanodrop">Nanodrop Protocol</div><div class="protocolContent"></html>{{:Team:University_College_London/Protocols/Nanodrop}}<html></div></html>
+Results: Nanodrop concentrations are presented in the table below. All are low except for the backbone containing kanamycin. We concluded that PCR for these backbones needs to be repeated
+{| class="wikitable"
+|-
+! BioBrick !! λ260 !! λ 280
+|-
+| PSB1A3 (ng/μl) || 27.3  || 26.6
+|-
+| PSB1C3 (ng/μl) || 23 || 24.4
+|-
+| PSB1K3 (ng/μl) ||66.5  || 87
+|-
+|}
+'''Conclusion:''' The PCR for PSB1A3 and PSB1C3 need to be repeated.
 <html>
 </div><div class="experiment"></div>
 <img id="8-3" src="https://static.igem.org/mediawiki/2012/f/f0/Ucl2012-labbook-graph8-3.png" /><div class="experimentContent">
 </html>
-== 8-3 ==
+==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 W3110 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.
+{{:Team:University_College_London/templates/pictureinsert|title=gel2|url=images/8/88/UCLigem2012.Salt_tolerance.png}}
+{{:Team:University_College_London/templates/pictureinsert|title=gel2|url=images/9/95/UCLigem2012.Salt_tolerance_1.png}}
+'''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.
 <html>
 </div><div class="experiment"></div>
 <img id="8-4" src="https://static.igem.org/mediawiki/2012/0/00/Ucl2012-labbook-graph8-4.png" /><div class="experimentContent">
 </html>
-== 8-4 ==
+== 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.
+'''Method'''
+==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) and B (Pcst + BB0034) constructs.
+'''Result:''' Observations showed that 3A was successful for the construct A, as shown in the image below. However there was no growth assembled pCST+BBa_B0034 (construct B). The following table summarises the results.
+{{:Team:University_College_London/templates/pictureinsert|title=Picture 1|url=images/9/94/Ucligem8.4.png}}
+'''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).
+'''Method'''
+<html><div class="protocol protocol-ColPic">Picking Colonies Protocol</div><div class="protocolContent"></html>{{:Team:University_College_London/Protocols/ColPic}}<html></div></html>
+== Friday 3.8.12 ==
+'''Aim:'''Check the results of colony picking.
+'''Results:''' There was growth. Next we can proceed to run a gel and a nanodrop to detect the presence of the correct bands, and whether we have a useable concentration.
 <html>
 </div><div class="experiment"></div>
 <img id="8-5" src="https://static.igem.org/mediawiki/2012/b/bd/Ucl2012-labbook-graph8-5.png" /><div class="experimentContent">
 </html>
-== 8-5 ==
+== Thursday 2.8.12 ==
+'''Aim - Transformation of alternative BioBricks:''' We have colony picked the BBa_J23119 constitutive promoter and the BBa_B0034 Ribosome Binding Site twice (Expt 6.3 & Expt 7,3), and gained a reasonable plasmid concentration. However the concentration was not high enough to be able to detect the product on the gel. This concerns us, because although we have detected the correct plasmid backbone, we are worried that we lack evidence that the transformation was successful. One option is to attempt several other BioBricks with the same function, in order to increase the chances of generating a product on the gel - and confirming that we definitely have a BioBricks with the required function.It would also increase the chance of generating successful ligations if we can reach a higher concentration of plasmid. We will also be reattempting the transformation of BBa_I750016 which failed several attempts at colony picking.
+'''Method:'''
+<html><div class="protocol protocol-Transformation">Transformation Protocol 1</div><div class="protocolContent"></html>{{:Team:University_College_London/Protocols/Transformation1}}<html></div></html>
+'''Step 1 – Thawing Cells:''' Use W3110 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
+{| class="wikitable"
+|-
+!  colspan="2" |   !! Function !! Module
+|-
+| rowspan="4" |BioBrick|| BBa_J23100 || Constitutive Promoter || All
+|-
+| BBa_J23106 || Constitutive Promoter || All
+|-
+| BBa_B0030 || Ribosome Binding Site || All
+|-
+| BBa_I750016 || Ribosome Binding Site || All
+|-
+| rowspan="2"|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).
+{| class="wikitable"
+|-
+! colspan="2" |  Samples !! Volume Inoculated !! Antibiotic in Gel (ug/ml)
+|-
+| rowspan="8" |BioBrick ||rowspan="2" | BBa_J23100  || 10ul || rowspan="8" | Ampicillin(50ug/ml)
+|-
+| 90ul
+|-
+| rowspan="2" | BBa_J23106  || 10ul
+|-
+| 90ul
+|-
+| rowspan="2" | BBa_B0030 || 10ul
+|-
+| 90ul
+|-
+| rowspan="2" | BBa_I750016 || 10ul
+|-
+| 90ul
+|-
+| rowspan="2" | Control || Positive || 36ul || No Antibiotic
+|-
+| Negative (No BioBrick)|| 36ul ||  1x Ampicillin(50ug/ml)
+|-
+|}
+== Friday 3.8.12 ==
+'''Aim - Check results of Transformation:''' The table below indicates whether there was growth on the Agar Plates after Transformation. Included below are images of the Agar Plates for each BioBrick.
+{| class="wikitable"
+|-
+! colspan="2" |  Samples !! Volume Inoculated !! Colony Formation
+|-
+| rowspan="8" |BioBrick ||rowspan="2" | BBa_J23100  || 10ul || Yes
+|-
+| 90ul || Yes
+|-
+| rowspan="2" | BBa_J23106  || 10ul || Yes
+|-
+| 90ul || Yes
+|-
+| rowspan="2" | BBa_B0030  || 10ul || Yes
+|-
+| 90ul || Yes
+|-
+| rowspan="2" | BBa_I750016  || 10ul || Yes
+|-
+| 90ul || Yes
+|-
+| rowspan="2" | Control || Positive (Contains BioBrick BBa_C0040)|| 36ul || Yes
+|-
+| Negative (No BioBrick)|| 36ul || Yes
+|-
+|}
+{{:Team:University_College_London/templates/pictureinsert|title=Picture 1|url=images/b/b3/Ucligem20128.5.png}}
+'''Conclusion:''' We had growth for all of our BioBricks, but also for the negative control which indicates there was contamination. Given we have had several issues with negative control recently, we decided we would not let the contamination delay the project. We will risk colony picking with the hope that we have some correctly transformed colonies. This will be commenced next week. It is worth noting that for Constitutive Promoter BBa_J23106 and for the Gas Vesicle Cluster BBa_I750016 there was a continuous film of growth. This suggests to us that the agar was not selective enough.
 <html>
 </div><div class="experiment"></div></html>
 {{:Team:University_College_London/templates/foot}}

Team:University College London/LabBook/Week8

From 2012.igem.org

Latest revision as of 01:23, 27 September 2012

Contents

Monday 30.6.12

Monday 30.7.12

Tuesday 31.7.12

Wednesday 1.8.12

Monday 30.7.12

Tuesday 31.8.12

Wednesday 1.8.12

Tuesday 31.7.12

Wednesday 1.8.12

Friday 3.8.12

Tuesday 31.7.12

Wednesday 1.8.12

Wednesday 1.8.12

Thursday 2.8.12

Friday 3.8.12

Thursday 2.8.12

Friday 3.8.12