Team:University College London/LabBook/Week7

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Contents

Monday 23.7.12

Aim - Transformation of TetR BBa_C0040 BioBrick

Enzyme Digest

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

Step 3 – Addition of BioBrick: To a 2ml eppendorf, add 1ul of the following BioBricks. Note: we have changed the protocol for our positive control. Previously it contained no BioBrick, but it has been recommended to us that we transform our positive control such that there is one for each BioBrick – this will tell us if the BioBrick has in any way affected cell viability. This will be used from this point onwards. Include an extra tube as a negative control, with no BioBrick added

Samples Function Module
BioBrick BBa_C0040 Tetracycline Repressor Buoyancy
Control Positive (Contains BioBrick BBa_C0040)
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.

Samples Volume Inoculated Antibiotic in Gel (ug/ml)
BioBrick BBa_C0040 10ul Ampicillin(50ug/ml)
90ul
Control Positive (Contains BioBrick BBa_C0040) 36ul No Antibiotic
Negative (No BioBrick) 36ul 2x Ampicillin(50ug/ml)

Tuesday 24.7.12

Aim - Results of Transformation

Result: The table below indicates that there was growth for this transformation.

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


Monday 23.7.12

Aim - Repeat Restriction Digest for BioBricks in Expt 4.1 and 5.1, where the gel previously was inconclusive: This is intended to diagnose whether the correct plasmid had been transformed into our bacteria, by measuring the size of the digested products against a DNA ladder. In previous gel attempts, K540000 has produced a band of the correct size, but we are repeating it because of the presence of other unexpected bands, which we expect are contaminants from the reaction. A previous restriction digest of BBa_I13522 has failed to produce a band of the correct size, so we are repeating the digest before considering another transformation. For the same reason, we are repeating the digest of BBaK398108, which produced bands of incorrect size, which is suggestive of contamination.


(LOGO) Restriction Digest


Step 2: Set up as follows

Samples Recipe Enzymes
BioBricks BBa_ K540000 (Expt 4.1) Digested Xbar 1 & Spe1
BBa_ I13522 (Expt 4.1) Digested Xbar 1 & Spe1
BBa_ K398108 (Expt 5.1) Digested Xbar 1 & Spe1
BBa_ KI32003 (Expt 5.1) Digested Xbar 1 & Spe1


(LOGO) Gel Electrophoresis


Results: The image below shows a 1% Agarose Gel of an Analytical Restriction Enzyme Digest for Expt 7.2. Visible in Lane 1 is a product of the correct size for the BBa_K540000 insert (3123bp), as indicated by A. Also shown is a correct sized product for the backbone PSB1C3 (2070bp), as indicated by B. Lane 2 shows a product corresponding to the size of the BBa_I13522 insert (937bp) as indicated by C. Also present is a product corresponding to the size of the PSB1A3 backbone (2155bp) as indicated by D. Lane 3 has a product corresponding to the expected size of insert BBa_K398108 (844bp) as indicated by E, and a product of the expected size for the plasmid backbone PSB1C3 (2070bp) as indicated by F. Lane 4 shows no products, where we would expect a product of size 1849bp (indicated by G) and a product of 2155 (indicated by H).



Conclusion: Plasmids Bba_k540000 (3123), Bba_I13522 (937) and Bba_K391108 (844) have produced bands of the correct size. The band at 2000 is the plasmid backbone. The failure of Bba_K123003 to produce a band is not of great concern, as we do not expect we will require this BioBrick. However, we may repeat it at a later date.


Tuesday 24.7.12

Aim - Colony Picking from unsuccessful colonies for Expt 7.1 and 6.3


(LOGO) Picking Colonies


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 Number of Falcons Broth Antibiotic
Biobrick BBa_C0040 3 Lysogeny Broth (5ml) Ampicillin(50ug/ml)
BBa_I750016 4
BBa_B0034 5
BBa_B0015 3
BBa_J23119 2

Wednesday 23.7.12

Aim - Results from Colony Picking


Results:

Samples Number of Falcons Growth
BioBrick BBa_C0040 3 None
BBa_I750016 4 None
BBa_B0034 5 Growth in one
BBa_B0015 3 Growth
BBa_J23119 2 Growth

Conclusion: We are beginning to consider other possibilities for the failure of Colony Picking in Expt 7.1 and Expt 6.3. Foremost, we believe it may be due to the addition of Ampicillin to the Agar before it has cooled sufficiently, leading to degradation of the antibiotic. This would reduce the selective pressure, and allow growth of colonies that are not Ampicillin-resistant. Subsequent inoculation into Amipicillin positive LB broth leads to failed growth. Those BioBricks that have worked will undergo Miniprep, Restriction Enzyme Digests and Nanodrop.

Method

Miniprep: (LOGO) Miniprep Protocol 1 (Qiagen)


Step 1: Set up an eppendorf as follows

Step ?: Used 2mls

Step ?: Step ? had to be missed because we realised too late we had too little PE? buffer remaining.


Restriction Digest: (LOGO) Restriction Digest Protocol 1


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 Xcar1/Spe1 digested sample be set up for each BioBrick. et up Eppendorfs as follows


Samples Recipe Enzymes
BioBrick BBa_B0034 Digested Plasmid Xbar 1 & Spe1
Undigested Plasmid (Control) None
BBa_B0015 Digested Plasmid Xbar 1 & Spe1
Undigested Plasmid (Control) None
BBa_B0034 Digested Plasmid Xbar 1 & Spe1
Undigested Plasmid (Control) None

(LOGO) Gel Electrophoresis


Results: The image below shows a 1% Agarose Gel of an Analytical Restriction Enzyme Digest for Expt 7.2. Visible in Lane 1 is a band corresponding to the Plasmid Backbone PSB1A2 (2079bp) as show by A, but it is not possible to detect our BBa_J23119 insert on this gel, as it is just 35bp long. Lane 2 shows the uncut plasmid for BBa_J23119 (shown by B) but the size difference between A and B appears to large for Bba_J23119 to have been the insert. This will require further investigation. Lane 3 shows a product 129bp long (indicated by C) which is the correct size for our insert BBa_B0015. A stronger band is also visible, indicated by D, which corresponds to the plasmid backbone PSB1AK3 (3189bp). Lane 4 displays a product which is somewhat larger than the expected size for the uncut plasmid (3318bp) as indicated by E. Lane 5 displays the correct product for the PSB1A2 backbone (2079bp) as indicated by F, but the extremely small size of the Bba_B0034 insert (12bp) means that it cannot be detect on this gel. Lane 6 indicates a band larger than expected, and does not correspond to the size of the uncut plasmid (2091bp) as shown by G.


(INSERT PIC 2)

Conclusion: The BBa_B0015 transformation was a success. However, with regard BBa_J23119, we feel that the difference in the product size of A (PSB1A2 plasmid backbone) and B (uncut plasmid) raises concerns as to whether the insert could be as short as 35bp. However, we must also consider the secondary effects had by the conformation of an uncut plasmid on its migration through the gel. It is possible this is sufficient to misplace the plasmid, such that its position does not represent its size. Even without this possibility, however, it would not be possible to determine whether there was a 35bp difference on such a crude scale of ladder. Instead we will reattempt the Analysis against a 25bp ladder, with the intention of detecting the insert. (See Expt 8.1). We have similar concerns for BBa_B0034, and so this too will be run against a 25bp ladder.

Nanodrop:

(LOGO) Nanodrop

BioBrick λ260 λ 280
BBa_J23119 (ng/μl) 77.6 71.8
BBa_B0034 (ng/μl) 70 71.2
BBa_B0015 (ng/μl) 128.9 128

Thursday 26.7.12

Aim - Transformation of previously failed BioBricks: BBa_I750016 was previously grown on an Agar Plate (Expt 6.3) but failed to produce growth after colony picking. BBa_C0040 was previously grown on an Agar Plate (Expt 7.1) but also failed to produce growth after colony picking. R0040 failed to produce colonies in Expt 5.1.


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_I750016 Gas Vesicle Polycistronic Cluster Buoyancy
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_I750016 10ul Ampicillin(50ug/ml)
90ul
BBa_C0040 10ul Ampicillin(50ug/ml)
90ul
BBa_R0040 10ul Ampicillin(50ug/ml)
90ul
Control Positive (Contains BioBrick BBa_C0040) 36ul No Antibiotic
Negative (No BioBrick) 36ul 1x Ampicillin(50ug/ml)


Friday 27.7.12

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_I750016 10ul No
90ul No
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

(Insert Pic 3)

Conclusion: Given there was growth on the negative control we are wary of our results. We feel the contamination was not a result of any of our materials, but rather because we were urged to do our colony transfers outside of the sterile hood. This was suggested, because as the inoculation occurs onto an antibiotic positive agar, it should be possible to do the transfer in a non sterile environment. However, this is the first occasion we have had difficulty with out negative control, and so we feel the lack of sterile environment was the cause. Moreover, the pattern of spread on the agar is suggestive of a contaminant. A similar pattern is seen on our BBa_C0040 plates, and so it is likely we will repeat these. A very different pattern was observed on the BBa_I750016 agars, and so we will carry out an analytical digest to determine whether it has been transformed correctly.