Team:Newcastle/Project/Labwork/Week1

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=Introductory Lab Sessions=
=Introductory Lab Sessions=
[[Image:Team-Newcastle iGem 2009 - Lab 03-07-09 no 1.JPG|thumb|right| Jane and Jess calculating serial dilutions]]
[[Image:Team-Newcastle iGem 2009 - Lab 03-07-09 no 1.JPG|thumb|right| Jane and Jess calculating serial dilutions]]
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==[https://2009.igem.org/Team:Newcastle/Project/Labwork/Week1/Week1 Week 1]==
 
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==Lab rules==
 
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* Always use the distilled water from the green tap
 
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* Always label things at the lab with the red tape. Put the initials, the lab name (PA), date and its contents
 
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* For anything which needs to be autoclaved, label, stick the autoclave tape, and send for autoclaving
 
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* Clean the bench before and after the work with ethanol
 
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* Dry the cases before using them
 
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* Keep a bottle/beaker of distilled water to use on the table
 
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* No actual work is allowed after 5 or 6 pm - if the autoclaver is needed, the experiments requiring it should be presented to the operator by 4pm
 
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=Lab Sessions - Week 2 (6th July - 10th July)=
 
<br>
<br>
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==Lab session: 6th July==
 
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[[Image:Team Newcastle iGem 2009 06-07-09 no 2.JPG|thumb|right| Goksel rehydrating the BioBricks, which are in dry DNA form]]
 
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List of bricks for the day
 
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#[http://partsregistry.org/Part:BBa_J04450 BBa_J04450] (plasmid:pSB1AK3). Location: Plate 1, Well 13A
 
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#[http://partsregistry.org/Part:BBa_I13522 BBa_I13522] (plasmid:pSB1AK3). Location: Plate 2, Well 8A
 
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#[http://partsregistry.org/Part:BBa_J04450 BBa_J04450] (plasmid:pSB1AT3). Location: Plate 1, Well 15A
 
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===Preparation of the tubes===
 
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* We got 3 tubes containing cells from the freezer at 80C and placed them into the ice and waited for 30 minutes
 
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* We prepared 6 tubes (3 tubes for controls and 3 tubes for the plasmids) for the bricks
 
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* We added 40ul of cells to each of the six tubes
 
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* We added 3ul of DNA for the plasmid tubes and 3ul of water for the control tubes
 
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* We added 900ul of LB for each of the six tubes
 
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* We then placed the tubes in the shaking incubator for an hour.
 
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===Plating out===
 
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[[Image:Team Newcastle iGem 2009 06-07-09 no 7.JPG|thumb|right| James extracting portion of diluted solution to be placed on plates]]
 
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[[Image:Team Newcastle iGem 2009 06-07-09 no 14.JPG|thumb|right| Hanny spreading solutions on plate using sterile glass beads]]
 
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We prepared 9 plates with full strength (undiluted) and 9 plates with diluted tube content.
 
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;Plates with undiluted content
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*[https://2009.igem.org/Team:Newcastle/Project/Labwork/Week1/Week1 Introductory Lab Sessions] (30th June - 3rd July 2009)
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*The first three plates having LB + Amp were used as the negative control. Hence the cells not having amp. resistance would not grow on these plates.
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* The next three plates with LB content were used as the positive control. Cells without amp. resistanec would still grow on these plates.
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* The other three plates were used for the plasmids.
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{| class="wikitable" border="1"
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*[https://2009.igem.org/Team:Newcastle/Project/Labwork/Week1/Week2 Introductory Lab Sessions] (6th July - 10th July 2009)
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|-
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| LB + Amp, (-) control 1
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| LB, (+) control 1
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| LB + Amp, Transformation Plasmid 1
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|-
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| LB + Amp, (-) control 2
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| LB, (+) control 2
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| LB + Amp, Transformation Plasmid 2
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|-
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| LB + Amp, (-) control 3
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| LB, (+) control 3
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| LB + Amp, Transformation Plasmid 3
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|-
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|}
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*[https://2009.igem.org/Team:Newcastle/Project/Labwork/Week1/Week3 Introductory Lab Sessions] (14th July - 17th July 2009)
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;Plates with diluted content
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* For the diluted ones we used 900ul of LB and 100ul of DNA from the corresponding tubes.
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*[https://2009.igem.org/Team:Newcastle/Project/Labwork/Week1/Week4 Introductory Lab Sessions] (23rd July - 24th July 2009)
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* The same set of 9 plates were prepared for the diluted content.
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<br>
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==Lab Session:7th July==
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====Experiment summary so far...====
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Yesterday, we attempted to transform cultures with BioBricks. We inserted the three BioBricks into competent <i>E.coli</i> cells grown in the previous lab session (via plasmids) and grew them overnight. The cells were spread on plates in both undiluted and diluted forms. The diluted solution of competent <i>E.coli</i> cells were spread onto three different plates - a negative control plate, a positive control plate and a plasmid plate (see table above). Today we reviewed the cultures which grew on the plates and prepared them for plasmid extraction and purification - this will form tomorrow's experiment.
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<br>
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====Observations====
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Firstly, none of the <i>E.coli</i> cells mixed with BioBrick 1 (in the presence of competence inducers) took up the DNA - they didn't appear on the diluted or undiluted plates, which were lined with ampicillin. However the <i>E.coli</i> cells which had been mixed with BioBricks 2 and 3 did take up the DNA as they were present in colonies on ampicillin-covered plates. There were two colonies of bacteria which had been transformed by BioBrick 2, and eight colonies transformed by BioBrick 3. These 10 cultures became the focus of the subsequent experiments.
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<br>
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====Preparation of Solutions====
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To prove that the DNA the <i>E.coli</i> cells had taken up really was the BioBrick added to the solution, we need to carry out the procedure of DNA electrophoresis with the aid of restriction enzymes. In order to carry out this step, we need to extract and purify the plasmids first (to be done tomorrow) via alkaline lysis - this requires three solutions, two of which were made today.
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<br>
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=====Solution 1=====
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According to Dr. Aldridge's protocol named "Phil’s mini method for Alkaline Lysis for Mini Prep", we need to add 'solution 1' (along with an RNAse) to resuspended transformed ''E.coli'' cells, tomorrow. Solution 1 requires the following ingredients at the following concentrations:
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    * 50 mM Glucose
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    * 25 mM Tris.Hcl (pH 8.0)
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    * 10 mM EDTA (pH 8.0)
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<b>This solution was made today in preparation for tomorrow's exercise.</b> We were told that the total volume should be 250ml. We were presented with pre-made solutions - 1M Tris HCl and 0.5M EDTA - and were told we needed to calculate the correct volumes of each of these solutions to be added to the beaker to give the desired molarity.
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<br>
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We were presented with glucose in it's solid form and asked to make a stock solution of 1M glucose; after which we were asked to calculate the amount to glucose solution to add to the beaker to make a molarity of 0.5M! The following equation helped:
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<br>
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Molecular Weight    x    Desired Volume (Litres)  x  Molarity  =  Weight (grams)
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* '''Making the glucose stock solution'''
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** molecular weight of glucose = 180.2
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** desired volume of stock solution = 500ml
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** desired molarity of stock = 1M
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Put these values into the equation above gives the amount of glucose solid needed for 500ml of water...
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<br>
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180.2  x  0.5l  x  1M  =  90.1g
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We added to 90.1 grams of glucose to 0.5 litres to get a 1M stock solution.
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*'''Adding the solutions to the beaker'''
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** 25ml of 1M glucose stock solution was transferred to beaker (to make 50mM)
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** 6.25ml of 1M Tris.HCl was transferred to the same beaker (to make 25mM)
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** 5ml of 0.5M EDTA also added (to make 10mM)
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** H2O made the rest of the solution
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<b> These values are derived from the above equation</b>
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<br>
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=====Solution 3=====
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We also needed to make solution 3 for tomorrow's practical. This involved the following ingredients:
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* 5M Potassium acetate: 60ml
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    * glacial acetic acid: 11.5 ml
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    * H2O: 28.5 ml
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The glacial acetic acid and the water could be added to the 'Solution 3' beaker immediately, but calculations had to be carried out for the 5M potassium acetate to determine the mass of the solid. We decided to make a 200ml stock of 5M solution.
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<br>
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* The molecular weight of potassium acetate = 98.15
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* The desired volume of this solution = 0.2 litres
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* The desired molarity = 5M
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<br>
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Put this into the equation:
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<br>
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Molecular Weight x Desired Volume (Litres) x Molarity = Weight (grams)
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<br>
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    98.15      x        0.2l      x  5M    = 98.15 grams
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<br>
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We then made up the solution and added it to the other ingredients, according to the protocol.
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<br>
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====Growing the transformed cultures====
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[[Image:Team Newcastle iGem 2009 07-07-09 no 1.JPG|thumb|right|Jess obtaining ''E.coli'' from a single colony of transformed cells]]
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With the solutions prepared and sent to the autoclaver, we had the task of taking each of the 10 colonies of transformed cells and placing them into 10 tubes containing LB. We labelled the tubes 1-10 and added the colonies to the tubes as follows:
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* Tube 1 = BioBrick 2 - colony 1
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* Tube 2 = BioBrick 2 - colony 2
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* Tube 3 = BioBrick 3 - colony 1
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* Tube 4 = BioBrick 3 - colony 2
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* Tube 5 = BioBrick 3 - colony 3
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* Tube 6 = BioBrick 3 - colony 4
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* Tube 7 = BioBrick 3 - colony 5
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* Tube 8 = BioBrick 3 - colony 6
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* Tube 9 = BioBrick 3 - colony 7
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* Tube 10 = BioBrick 3 - colony 8
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<br>
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* BioBrick 2 = '''[http://partsregistry.org/Part:BBa_I13522 BBa_I13522]''' in the '''pSB1AK3''' plasmid
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* BioBrick 3 = '''[http://partsregistry.org/Part:BBa_J04450 BBa_J04450]''' in the '''pSB1AT3''' plasmid
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<br>
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We then left the cultures to grow overnight.
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==Lab session: 8th July==
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[[Image:Team Newcastle iGem 2009 08-07-09 no 12.JPG|thumb|right| Goksel has every right to look optimistic! The experiment went according to plan]]
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====Experiment Recap====
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In summary, our team have attempted to transform ''E.coli'' with three BioBricks - '''[http://partsregistry.org/Part:BBa_J04450 BBa_J04450]''' (with plasmid '''pSB1AK3'''), '''[http://partsregistry.org/Part:BBa_I13522 BBa_I13522]''' (with plasmid '''pSB1AK3''') and '''[http://partsregistry.org/Part:BBa_J04450 BBa_J04450]''' (with plasmid '''PSB1AT3'''). The cells were cultured on ampicillin-covered agar plates overnight after being treated with both the BioBrick DNA and the competence inducer calcium chloride. Only 10 colonies emerged on the ampicillin-covered plates - '''2 cultures transformed with BioBrick BBa_I13522 and 8 cultures transformed with BioBrick BBa_J04450'''.
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These cultures were then placed in some nutrient broth and left overnight to replicate and amplify the plasmid concentration. Today, we will process these cultures of cells by extracting and purifying the plasmid DNA - this will then be used in DNA electrophoresis to confirm that the DNA inherited by the ampicillin resistant bacteria are the BioBricks we inserted.
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<br>
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====Observations====
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<center>[[Image:Team Newcastle iGem 2009 08-07-09 no 8.JPG|350px| The 10 transformed cultures of ''E.coli'' - Tubes 1 and 2 contain BioBrick BBa_I13522 (with plasmid pSB1AK3) which encodes '''GFP'''; Tubes 3-10 contain BioBrick BBa_J04450 (with plasmid PSB1AT3) which encodes '''RFP''']]</center>
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<br>
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The above image shows the appearance of the 10 tubes after a night of incubation in the shaker. Tubes 1 and 2 showed yellow, translucent cultures, whereas tubes 3-10 showed red, translucent cultures. This is because tubes 1 and 2 contain ''E.coli'' transformed by the biobrick '''[http://partsregistry.org/Part:BBa_I13522 BBa_I13522]''' (with plasmid pSB1AK3) which '''encodes Green Fluorescence Protein (GFP)''' whereas tubes 3-10 carry the BioBrick '''[http://partsregistry.org/Part:BBa_J04450 BBa_J04450]''' (with plasmid PSB1AT3), which '''encodes RFP (Red Fluorescence Protein)'''.
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<br>
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====Today's Procedure====
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[[Image:Team Newcastle iGem 2009 08-07-09 no 13.JPG|thumb|right|Goksel, James and Mathew working on procedure]]
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# spin and resuspend the 10 cultures in 300ul of Sol.I+RNase
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# Add 600 ul '''fresh''' Sol.II
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# 5 min RT then add 250 ul Sol III
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# Centrifuge for 20 mins
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# Put 1ml supernatant into new tube and add 600 ul Isopropanol
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# Spin for 15 min and aspirate
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# Add 500 ul 70% ethanol and spin for 5 min
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# Aspirate and speed vac resuspending in 50 ul water
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<br>
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=====Making up Solution II=====
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The procedure states that Solution II should be made 'fresh' so we had to make it today and before we carried out the rest of the procedure. The solution is made up of the following:
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* 2ml NaOH (1M)
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* 1ml 10%SDS
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* 7 ml water
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Because the volumes and the molarities of the recipe were given and because the solutions of NaOH and SDS were made up already, there was no need for calculations.
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=====Carrying out the rest of the procedure=====
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[[Image:Team Newcastle iGem 2009 08-07-09 no 9.JPG|thumb|right|James removing the supernatant from the Eppendorf tubes]]
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[[Image:Team Newcastle iGem 2009 08-07-09 no 10.JPG|thumb|right|Goksel discards the supernatant extracted from the freshly centrifuged tubes]]
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* The procedure demanded us to centrifuge 3ml (at least) of the 10 cultures. However the Eppendorf tubes that are placed into the centrifuge dont't have that capacity. So we pipetted 1.5ml of the 10 cultures into 10 Eppendorf tubes, spun them, removed the supernatant and added a further 1.5ml of the cultures to the 10 tubes. This was spun and the supernatant removed.
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<br>
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* The 10 pellets were then each resuspended in 300 microlitres of Solution I. Prior to this, we added 150 microlitres of RNAse A to the bottle containing our premade Solution I.
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<br>
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* 600 microlitres of the newly made Solution II was added to each of the 10 tubes. The tubes were then left (sealed, of course) in the rack for 5 mins.
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<br>
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* 250 microlitres of Solution III was added to each of the 10 tubes and the resulting solution centrifuged for 20 minutes. The resulting solution could be seen as a cloudy white precipitate of chromosomal DNA and protein. Instead of removing the supernatant and disposing of it, once the spinning had stopped we extracted 1ml of supernatant and placed them into 10 new Eppendorf Tubes (labelled '1S', '2S', '3S',...etc). The tubes containing the pellets were then discarded.
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<br>
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* To the 10 tubes containing supernatant, 600 microlitres of prepared isopropanol was added. These tubes were then placed in the centrifuge for a further 15 mins.
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<br>
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* The resulting supernatants for the 10 tubes were then removed and any droplets of solution were carefully removed by a vacuum pump (without disrupting the DNA which had gathered on one side of the Eppendorf tubes). To this, 500 microlitres of ethanol (at 70%) was added.
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<br>
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* Making 70% ethanol:
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** 35 ml of ethanol added to beaker
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** 15 ml of water added to beaker
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<br>
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* The resulting solution was then spun in the centrifuge for 5 minutes.
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<br>
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*The supernatant was removed and the remaining droplets of solution removed without disrupting the transparent pellet the plasmids had formed. Once the plasmid pellet was dried of all the solution, it was resuspended in 50 microlitres of water. The tubes were then lightly centrifuged (for a few seconds) to make sure all of the water, along with the DNA, had congregated at the bottom of the tubes.
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<br>
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====Conclusion====
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We are now ready to carry out DNA electrophoresis on the 10 samples, which will happen tomorrow.
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<br>
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==Lab session: 9th July==
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====Experiment Recap====
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[[Image:Team Newcastle iGem 2009 08-07-09 no 8.JPG|150px|thumb|right|10 colonies of transformed ''E.coli'' cells - 2 containing GFP, 8 containing RFP]]
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Up to this point, we have successfully isolated and cultured single colonies of E.coli cells. With these micro-organisms, we were able to induce competence and had the opportunity to transform them with the following BioBricks:
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* [http://partsregistry.org/Part:BBa_J04450 BBa_J04450] with plasmid pSB1AK3
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* [http://partsregistry.org/Part:BBa_I13522 BBa_I13522] with plasmid pSB1AK3
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* [http://partsregistry.org/Part:BBa_J04450 BBa_J04450] with plasmid pSB1AT3
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<br>
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So far, we are convinced that 2 colonies of our cultured ''E.coli'' cells have become transformed by BioBrick BBa_I13522 (with plasmid pSB1AK3) which encodes GFP and that 10 colonies of our cultured ''E.coli'' cells have been transformed by BioBrick BBa_J04450 (with plasmid pSB1AT3), which leads to the production of RFP. This can be seen visually in the tubes; see image in the previous experiment under the heading '[https://2009.igem.org/Team:Newcastle/Project/Labwork/Week1#Observations_2 Observations]'.
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However visual representations may not always available and in these scenarios, it is difficult to tell whether the ''E.coli'' have become transformed by the given BioBrick unless DNA analysis is conducted. Even though the cells have survived the negative selection process (i.e. antibiotic exposure), it doesn't mean that they all possess the BioBrick - they could have received that resistance through other means.
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====Aim of Today's Experiment====
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[[Image:Team Newcastle iGem 2009 09-07-09 no 17.JPG|200px|thumb|right| The gel displaying the three marker bands - the cyan, dark blue and yellow marker bands]]
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We need to get ourselves familiarised with conducting DNA gel electrophoresis. We shall be doing this by preparing and then inserting the plasmid DNA (extracted from the 10 colonies of cells) into the gel. At this stage, the plasmids will be inserted as uncut objects because no restriction enzymes have been added yet (the analysis of cut plasmids comes at a later stage). Thus we won't be proving that it is the BioBrick which is causing the bacteria to display the properties seen so far. We will be proving that the bacteria have taken up plasmids.
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This procedure will see the team:
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* Prepare the DNA electrophoresis equipment by preparing the gel, administering buffer and ethidium bromide, etc
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* Prepare the plasmid DNA for gel electrophoresis.
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* Apply the DNA to the gel and conduct electrophoresis
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* Capture an image of the gel using UV-scanner.
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<br>
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====Experiment procedures====
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[[Image:Team Newcastle iGem 2009 09-07-09 no 6.JPG|200px|thumb|right| Melting of the 0.8% agarose gel]]
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[[Image:Team Newcastle iGem 2009 09-07-09 no 8.JPG|200px|thumb|right|Pouring of TAE buffer + ethidium bromide]]
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The protocol we used for this practical can be found '[https://2009.igem.org/Team:Newcastle/Project/Labwork/MoreProtocols/DNAGel here].'
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<br>
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=====Preparing the gel=====
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* A premade stock solution of agarose gel (0.8% agarose) was taken and heated in microwave for 3 minutes under medium/high power initially. Short 30 second bursts were required to completely liquify the solution
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* Gel was then removed from microwave and left on bench to cool
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<br>
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=====Preparing the buffer=====
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* Premade solution of 1x TAE was used - took volume of this and added to flask
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* Added 8 microlitres to the TAE buffer
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<br>
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=====Preparing the gel electrophoresis equipment=====
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* Trays and comb were cleaned and provided for the team, thanks to the lab staff!
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* Inserted comb into groove nearest the edge of the tray and placed in proximity of the electrodes.
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<br>
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=====Pouring Buffer and Gel=====
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* Placed the 1x TAE buffer (plus ethidium bromide) into the tray (with gel comb inserted in groove nearest negative electrode)
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* Agarose gel solution placed into tray and bubbles removed using a pipette. After this, lid placed over the gel electrophoresis equipment.
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<br>
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=====Preparing DNA samples=====
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[[Image:Team Newcastle iGem 2009 09-07-09 no 12.JPG|200px|thumb|right| Jane extracting dye to be added to plasmid DNA]]
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* Removed 10 Eppendorf tubes (containing the plasmid DNA for the 10 colonies of ''E.coli'') from the freezer and defrosted them briefly. In the meantime, 10 fresh Eppendorf tubes were placed in rack and labelled '1-10' to match the 10 plasmid DNA Eppendorf tubes.
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* When defrosted, 9 microlitres of the plasmid DNA was transferred to new Eppendorf tubes. 1 microlitre of dye was then added to the new Eppendorf tubes. The tubes were then lightly centrifuged to make sure the dye and the plasmid DNA had mixed at the bottom of the tube.
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* An Eppendorf tube containing the standard DNA ladder and dye was added to the rack, in the first entry of the row with the 'DNA + dye' tubes following afterwards (in order of 1-10). This was then taken over to the gel electrophoresis tray.
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<br>
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=====Loading the samples and carrying out electrophoresis=====
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[[Image:Team Newcastle iGem 2009 09-07-09 no 16.JPG|200px|thumb|right| Jess carefully pipetting the plasmid DNA (plus dye) into the wells]]
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* Made sure agarose gel had completely set. After confirmation of setting, the gel comb was removed revealing wells in which to put the DNA into.
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* Making sure that the pipette was pointed vertically over the well and that the pipette was hovering over (and not poking) the well, 10 microlitres of DNA was slowly and gently added to each well.
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** Well 1 = DNA Ladder
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** Well 2 = plasmid DNA from colony 1 - BBa_I13522 with plasmid pSB1AK3
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** Well 3 = plasmid DNA from colony 2 - BBa_I13522 with plasmid pSB1AK3
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** Well 4 = plasmid DNA from colony 3 - BBa_J04450 with plasmid pSB1AT3
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** Well 5 = plasmid DNA from colony 4 - BBa_J04450 with plasmid pSB1AT3
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** Well 6 = plasmid DNA from colony 5 - BBa_J04450 with plasmid pSB1AT3
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** Well 7 = plasmid DNA from colony 6 - BBa_J04450 with plasmid pSB1AT3
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** Well 8 = plasmid DNA from colony 7 - BBa_J04450 with plasmid pSB1AT3
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** Well 9 = plasmid DNA from colony 8 - BBa_J04450 with plasmid pSB1AT3
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** Well 10 = plasmid DNA from colony 9 - BBa_J04450 with plasmid pSB1AT3
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** Well 11 = plasmid DNA from colony 10 - BBa_J04450 with plasmid pSB1AT3
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* Lid was applied and electrodes positioned correctly (positive electrode at end furthest from wells and negative electrode positioned nearest wells). Power was switched on and voltage set to 150 volts. This process was allowed to run for 40 minutes.
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<br>
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====Observations and Results====
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* Once 40 minutes had expired, electrodes were switched off and lid removed. In the gel it could be seen that the DNA had streaked from the wells to the positive electrode. Three marker bands were visible, in order from nearest wells to nearest positive electrode - a cyan band, a dark blue band and a yellow band.
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* Gel removed from gel tray and taken to UV scanner. The following photograph was produced:
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[[Image:Team Newcastle igem 2009 Transformation1-9-7-09.jpg|600px|center]]
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<br>
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From this photograph it can be seen that apart from the ''E.coli'' cells located in colony 9, all the colonies that had survived the negative selection and displayed either a red or a green colour in solution contained plasmid DNA. This proves that most of the colonies had been transformed with a plasmid at least. The possibilities for colony 9 not revealing a plasmid DNA readout are as follows:
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* Colony 9 ''E.coli'' cells did not receive the plasmid DNA and acquired it's ampicillin resistance and colour properties elsewhere
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* The plasmid DNA (which appears as a transparent pellet in the previous experiment) was lost when the supernatant was tipped out.
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Now that Team:Newcastle has proved that the competent ''E.coli'' cells have taken up the plasmids, the next step will be to prove that the 'E.coli' have taken up the specific BioBricks that we attempted to transform them with. This will involve restriction enzymes and another DNA gel electrophoresis.
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Latest revision as of 16:59, 18 August 2009



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