Team:University College London/LabBook/Week5

From 2012.igem.org

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'''Step 2 - Inoculating Colonies into a Selective Broth:''' The table below indicates the volume of broth and the concentration of antibiotic used for the BioBrick.
'''Step 2 - Inoculating Colonies into a Selective Broth:''' The table below indicates the volume of broth and the concentration of antibiotic used for the BioBrick.
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 +
{| class="wikitable"
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|-
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! colspan="2" |  Samples !! Volume Inoculated !! Broth (ml)!! Antibiotic (ug/ml)
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|-
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| rowspan="16" |BioBrick ||rowspan="2" | BBa_ K123003 || 20ul ||rowspan="2" | Lysogeny Broth (5)|| rowspan="2" | Ampicillin(50ug/ml)
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|-
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| 200ul 
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|}
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 +
 +
== Wednesday (11.7.12) ==
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'''Aim 1 – Check Results of Picking Colonies of BBa_K398108.'''
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 +
'''Result:''' The broth was cloudly, indicating there has been sufficient growth of bacteria to proceed to purification (miniprep) and  an Analytical Restriction Digest to determine the presence of the correct BioBrick.
 +
 +
Method
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(LOGO) Miniprep Protocol 1 – Qiagen
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(LOGO) Restriction Digest
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 +
'''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 Spe1/Xba1 digested sample be set up for each BioBrick.
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{| class="wikitable"
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|-
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! colspan="2" |  Samples !! Recipe !!Enzyme Used!! Buffer
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|-
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| rowspan="16" |BioBrick ||rowspan="2" | BBa_K398108 || Digested Plasmid || Spe1 and Xba1|| rowspan="2" | 4
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|-
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| Undigested Plasmid  || None
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|}
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(LOGO) Gel Electrophoresis
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Results: In Lane 1 and 2 we expect a product equivalent to the size of the PSB1C3 plasmid backbone and BBa_K398108 insert (2914bp) long, indicated by the position of A. The absence of this band, and the presence of other bands indicates that this transformation was unsuccessful. In Lane 3 and 4 we would expect a product for the BBa_K398108 insert (844bp) as indicated by B, and a product for the PSB1C3 plasmid backbone (2070bp) as indicated by C.  Both products are absent, further supporting the failure of the transformation. In Lane 5 and 6 we would expect to see a similar product to Lanes 1 and 2, except for any secondary effect that the conformation of the uncut plasmid may have on its migration. Such a product was not found.
 +
 +
(Insert Gel PIC)
 +
 +
'''Conclusion:''' This transformation failed, or contamination has occurred since the colony picking.
 +
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(LOGO) Nanodrop
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{| class="wikitable"
 +
|-
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! BioBrick!! 260 !! 280
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|-
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| BBa_K398108 || 173.8 || 186.6
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|}
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 +
'''Aim 2 - Picking Colonies for BBa_K123003.''' With the exception of BBa_K398108, which has already been analysed, BBa_K123003 was the only other BioBrick to show growth in the original Expt 5.1 cohort. Colonies from the Agar Plate will be selected and cultured, with the intent of purifying the plasmid for detecting (Restriction Enzyme Digest and Gel Electrophoresis) the presence of the correct BioBrick.
 +
 +
'''Method'''
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(LOGO) Picking Colonies
 +
 +
'''Step 2 - Inoculating Colonies into a Selective Broth:''' The table below indicates the volume of broth and the concentration of antibiotic used for inoculating the BioBrick.

Revision as of 12:35, 1 August 2012

Contents

Monday (9.7.12)

Aim: Transformation of Key BioBricks

Method

(LOGO) Transformation Protocol 2

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_K123003 Oestrogen Receptor Detection
BBa_K123002 Oestrogen Response Element Detection
BBa_K398108 Salt Tolerance Cluster Salt Tolerance
BBa_J23100 Constitutive Promoter
BBa_J23107 Constitutive Promoter
BBa_R0040 TetR Repressible Promoter Buoyancy
BBa_J23106 Constitutive Promoter
BBa_B0034 Ribosome Binding Site (RBS) All
Control (No BioBricks)

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.



Samples Volume Inoculated Antibiotic in Gel (ug/ml)
BioBrick BBa_ K123003 20ul Ampicillin(50ug/ml)
200ul
BBa_ K123002 20ul
200ul
BBa_B0034 20ul
200ul
BBa_J23100 20ul
200ul
BBa_J23107 20ul
200ul
BBa_R0040 20ul
200ul
BBa_J23106 20ul
200ul
BBa_ K398108 20ul Chloramphenicol (25ug/ml)
200ul
Control Positive (No BioBrick) 36ul No Antibiotic
Negative (No BioBrick) 36ul 2x Ampicillin(50ug/ml)

1x Chloramphenicol (25ug/ml)

Tuesday (10.7.12)

Aim - Check results from Transformation

Results: The table below indicates whether or not there was growth on each plate. Included is an image demonstrating the growth noted for BBa_K123003, BBa_K398108, and the Positive Control

Samples Volume Inoculated Growth/No Growth
BioBrick BBa_ K123003 20ul Growth
200ul Growth
BBa_ K123002 20ul No Growth
200ul No Growth
BBa_B0034 20ul No Growth
200ul No Growth
BBa_J23100 20ul No Growth
200ul No Growth
BBa_J23107 10ul No Growth
2000ul No Growth
BBa_R0040 20ul No Growth
200ul No Growth
BBa_J23106 20ul No Growth
200ul No Growth
BBa_ K398108 20ul No Growth
200ul Growth
Control Positive (No BioBrick) 36ul Growth
Negative (No BioBrick) 36ul No Growth

Conclusion: Only BBa_K398108 and BBa_K123003 had successfully transformed. This is a serious setback, which suggests our cell competency is not as high as we first anticipated. Method

(LOGO) Picking Colonies

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

Samples Volume Inoculated Broth (ml) Antibiotic (ug/ml)
BioBrick BBa_ K123003 20ul Lysogeny Broth (5) Ampicillin(50ug/ml)
200ul


Wednesday (11.7.12)

Aim 1 – Check Results of Picking Colonies of BBa_K398108.

Result: The broth was cloudly, indicating there has been sufficient growth of bacteria to proceed to purification (miniprep) and an Analytical Restriction Digest to determine the presence of the correct BioBrick.

Method (LOGO) Miniprep Protocol 1 – Qiagen (LOGO) Restriction Digest

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 Spe1/Xba1 digested sample be set up for each BioBrick.

Samples Recipe Enzyme Used Buffer
BioBrick BBa_K398108 Digested Plasmid Spe1 and Xba1 4
Undigested Plasmid None

(LOGO) Gel Electrophoresis

Results: In Lane 1 and 2 we expect a product equivalent to the size of the PSB1C3 plasmid backbone and BBa_K398108 insert (2914bp) long, indicated by the position of A. The absence of this band, and the presence of other bands indicates that this transformation was unsuccessful. In Lane 3 and 4 we would expect a product for the BBa_K398108 insert (844bp) as indicated by B, and a product for the PSB1C3 plasmid backbone (2070bp) as indicated by C. Both products are absent, further supporting the failure of the transformation. In Lane 5 and 6 we would expect to see a similar product to Lanes 1 and 2, except for any secondary effect that the conformation of the uncut plasmid may have on its migration. Such a product was not found.

(Insert Gel PIC)

Conclusion: This transformation failed, or contamination has occurred since the colony picking.

(LOGO) Nanodrop

BioBrick 260 280
BBa_K398108 173.8 186.6

Aim 2 - Picking Colonies for BBa_K123003. With the exception of BBa_K398108, which has already been analysed, BBa_K123003 was the only other BioBrick to show growth in the original Expt 5.1 cohort. Colonies from the Agar Plate will be selected and cultured, with the intent of purifying the plasmid for detecting (Restriction Enzyme Digest and Gel Electrophoresis) the presence of the correct BioBrick.

Method (LOGO) Picking Colonies

Step 2 - Inoculating Colonies into a Selective Broth: The table below indicates the volume of broth and the concentration of antibiotic used for inoculating the BioBrick.


4.1

5.3

5.2

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