Team:Newcastle/Project/Labwork/Week1/Week2

=Introductory Lab Sessions Week 2= (6th July - 10th July 2009)

The second week initial planning, 6th July - 10th July
Extract and purify the plasmid DNA from the transformed colonies Carry out DNA gel electrophoresis using this protocol Prepare restriction digests of plasmid DNA (for 10 colonies) for gel electrophoresis (which will happen next week)
 * Monday
 * 1) Locate three BioBricks and rehydrate them:
 * 2) BBa_J04450 (plasmid:pSB1AK3). Location: Plate 1, Well 13A
 * 3) BBa_I13522 (plasmid:pSB1AK3). Location: Plate 2, Well 8A
 * 4) BBa_J04450 (plasmid:pSB1AT3). Location: Plate 1, Well 15A
 * 5) Use the 3 cell cultures prepared from previous experiment:
 * 6) Place each culture into 2 tubes each (6 tubes in total)
 * 7) Add 3 biobricks to 3 tubes of culture (one BioBrick in each tube)
 * 8) To the other 3, add water (these will be controls)
 * 9) Plate tubes over 18 plates
 * 10) 9 plates of undiluted content
 * 11) LB + Amp (Negative control)
 * 12) LB (Positive control)
 * 13) LB + Amp (for plasmids)
 * 14) 9 plates of diluted content
 * 15) LB + Amp (Negative control)
 * 16) LB (Positive control)
 * 17) LB + Amp (for 'water')
 * Tuesday
 * 1) Preparation of solutions for Wednesday's experiment (purification of plasmid DNA)
 * 2) Make solution I
 * 3) Prepare solution III
 * 4) Grow transformed cultures (if any)
 * 5) Remove colonies from agar plates (make sure it's the LB + AMP (plasmids) colonies for each of the three BioBricks)
 * 6) Grow them in tubes containing fresh LB nutrient broth.
 * Wednesday
 * 1) Make up solution II
 * 2) Carry out remainder of procedure:
 * 3) Add premade solutions I and II where needed
 * 4) Prepare some isopropanol
 * 5) Make up solution of 70% ethanol.
 * Thursday
 * 1) Set up DNA gel electrophoresis apparatus:
 * 2) Prepare the gel
 * 3) Prepare the buffer
 * 4) Prepare the gel trays
 * 5) Pour buffers and gel
 * 6) Prepare DNA samples:
 * 7) Extract 9 microlitres of plasmid DNA
 * 8) Add 1 microlitre of dye to plasmid DNA extract
 * 9) Carry out electrophoresis
 * 10) Load wells
 * 11) Apply voltage for 40 mins
 * 12) Photograph gel under UV scanner
 * Friday

Lab session: 6th July
List of bricks for the day
 * 1) BBa_J04450 (plasmid:pSB1AK3). Location: Plate 1, Well 13A
 * 2) BBa_I13522 (plasmid:pSB1AK3). Location: Plate 2, Well 8A
 * 3) BBa_J04450 (plasmid:pSB1AT3). Location: Plate 1, Well 15A

Preparation of the tubes

 * We got 3 tubes containing cells from the freezer at 80C and placed them into the ice and waited for 30 minutes
 * We prepared 6 tubes (3 tubes for controls and 3 tubes for the plasmids) for the bricks
 * We added 40ul of cells to each of the six tubes
 * We added 3ul of DNA for the plasmid tubes and 3ul of water for the control tubes
 * We added 900ul of LB for each of the six tubes
 * We then placed the tubes in the shaking incubator for an hour.

Plating out
We prepared 9 plates with full strength (undiluted) and 9 plates with diluted tube content.


 * Plates with undiluted content
 * 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.
 * The next three plates with LB content were used as the positive control. Cells without amp. resistanec would still grow on these plates.
 * The other three plates were used for the plasmids.


 * Plates with diluted content
 * For the diluted ones we used 900ul of LB and 100ul of DNA from the corresponding tubes.
 * The same set of 9 plates were prepared for the diluted content.

Experiment summary so far...
Yesterday, we attempted to transform cultures with BioBricks. We inserted the three BioBricks into competent E.coli 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 E.coli 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.

Observations
Firstly, none of the E.coli 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 E.coli 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.

Preparation of Solutions
To prove that the DNA the E.coli 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.

Solution 1
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: * 50 mM Glucose * 25 mM Tris.Hcl (pH 8.0) * 10 mM EDTA (pH 8.0)

This solution was made today in preparation for tomorrow's exercise. 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.

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:

Molecular Weight   x    Desired Volume (Litres)   x   Molarity   =   Weight (grams)


 * Making the glucose stock solution
 * molecular weight of glucose = 180.2
 * desired volume of stock solution = 500ml
 * desired molarity of stock = 1M

Put these values into the equation above gives the amount of glucose solid needed for 500ml of water...

180.2  x   0.5l   x   1M   =   90.1g

We added to 90.1 grams of glucose to 0.5 litres to get a 1M stock solution.

 These values are derived from the above equation
 * Adding the solutions to the beaker
 * 25ml of 1M glucose stock solution was transferred to beaker (to make 50mM)
 * 6.25ml of 1M Tris.HCl was transferred to the same beaker (to make 25mM)
 * 5ml of 0.5M EDTA also added (to make 10mM)
 * H2O made the rest of the solution

Solution 3
We also needed to make solution 3 for tomorrow's practical. This involved the following ingredients:

* 5M Potassium acetate: 60ml * glacial acetic acid: 11.5 ml   * H2O: 28.5 ml

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. Put this into the equation: Molecular Weight x Desired Volume (Litres) x Molarity = Weight (grams) 98.15      x        0.2l      x   5M    = 98.15 grams We then made up the solution and added it to the other ingredients, according to the protocol.
 * The molecular weight of potassium acetate = 98.15
 * The desired volume of this solution = 0.2 litres
 * The desired molarity = 5M

Growing the transformed cultures
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:


 * Tube 1 = BioBrick 2 - colony 1
 * Tube 2 = BioBrick 2 - colony 2
 * Tube 3 = BioBrick 3 - colony 1
 * Tube 4 = BioBrick 3 - colony 2
 * Tube 5 = BioBrick 3 - colony 3
 * Tube 6 = BioBrick 3 - colony 4
 * Tube 7 = BioBrick 3 - colony 5
 * Tube 8 = BioBrick 3 - colony 6
 * Tube 9 = BioBrick 3 - colony 7
 * Tube 10 = BioBrick 3 - colony 8

We then left the cultures to grow overnight.
 * BioBrick 2 = BBa_I13522 in the pSB1AK3 plasmid
 * BioBrick 3 = BBa_J04450 in the pSB1AT3 plasmid

Experiment Recap
In summary, our team have attempted to transform E.coli with three BioBricks - BBa_J04450 (with plasmid pSB1AK3), BBa_I13522 (with plasmid pSB1AK3) and 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.

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.

Observations


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 BBa_I13522 (with plasmid pSB1AK3) which encodes Green Fluorescence Protein (GFP) whereas tubes 3-10 carry the BioBrick BBa_J04450 (with plasmid PSB1AT3), which encodes RFP (Red Fluorescence Protein).

Today's Procedure

 * 1) spin and resuspend the 10 cultures in 300ul of Sol.I+RNase
 * 2) Add 600 ul fresh Sol.II
 * 3) 5 min RT then add 250 ul Sol III
 * 4) Centrifuge for 20 mins
 * 5) Put 1ml supernatant into new tube and add 600 ul Isopropanol
 * 6) Spin for 15 min and aspirate
 * 7) Add 500 ul 70% ethanol and spin for 5 min
 * 8) Aspirate and speed vac resuspending in 50 ul water

Making up Solution II
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:
 * 2ml NaOH (1M)
 * 1ml 10%SDS
 * 7 ml water

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.

Carrying out the rest of the procedure

 * 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.
 * 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.
 * 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.
 * 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.
 * 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.
 * 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.


 * Making 70% ethanol:
 * 35 ml of ethanol added to beaker
 * 15 ml of water added to beaker


 * The resulting solution was then spun in the centrifuge for 5 minutes.
 * 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.

Conclusion
We are now ready to carry out DNA electrophoresis on the 10 samples, which will happen tomorrow.

Experiment Recap
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:


 * BBa_J04450 with plasmid pSB1AK3
 * BBa_I13522 with plasmid pSB1AK3
 * BBa_J04450 with plasmid pSB1AT3

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 'Observations'.

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.

Aim of Today's Experiment
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.

This procedure will see the team:
 * Prepare the DNA electrophoresis equipment by preparing the gel, administering buffer and ethidium bromide, etc
 * Prepare the plasmid DNA for gel electrophoresis.
 * Apply the DNA to the gel and conduct electrophoresis
 * Capture an image of the gel using UV-scanner.

Experiment procedures
The protocol we used for this practical can be found 'here.'

Preparing the gel

 * 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


 * Gel was then removed from microwave and left on bench to cool

Preparing the buffer

 * Premade solution of 1x TAE was used - took volume of this and added to flask


 * Added 8 microlitres to the TAE buffer

Preparing the gel electrophoresis equipment

 * Trays and comb were cleaned and provided for the team, thanks to the lab staff!


 * Inserted comb into groove nearest the edge of the tray and placed in proximity of the electrodes.

Pouring Buffer and Gel

 * Placed the 1x TAE buffer (plus ethidium bromide) into the tray (with gel comb inserted in groove nearest negative electrode)


 * Agarose gel solution placed into tray and bubbles removed using a pipette. After this, lid placed over the gel electrophoresis equipment.

Preparing DNA samples

 * 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.


 * 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.


 * 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.

Loading the samples and carrying out electrophoresis

 * 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.


 * 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.
 * Well 1 = DNA Ladder
 * Well 2 = plasmid DNA from colony 1 - BBa_I13522 with plasmid pSB1AK3
 * Well 3 = plasmid DNA from colony 2 - BBa_I13522 with plasmid pSB1AK3
 * Well 4 = plasmid DNA from colony 3 - BBa_J04450 with plasmid pSB1AT3
 * Well 5 = plasmid DNA from colony 4 - BBa_J04450 with plasmid pSB1AT3
 * Well 6 = plasmid DNA from colony 5 - BBa_J04450 with plasmid pSB1AT3
 * Well 7 = plasmid DNA from colony 6 - BBa_J04450 with plasmid pSB1AT3
 * Well 8 = plasmid DNA from colony 7 - BBa_J04450 with plasmid pSB1AT3
 * Well 9 = plasmid DNA from colony 8 - BBa_J04450 with plasmid pSB1AT3
 * Well 10 = plasmid DNA from colony 9 - BBa_J04450 with plasmid pSB1AT3
 * Well 11 = plasmid DNA from colony 10 - BBa_J04450 with plasmid pSB1AT3


 * 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.

Observations and Results

 * 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.

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:
 * Gel removed from gel tray and taken to UV scanner. The following photograph was produced:

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.
 * Colony 9 E.coli cells did not receive the plasmid DNA and acquired it's ampicillin resistance and colour properties elsewhere
 * The plasmid DNA (which appears as a transparent pellet in the previous experiment) was lost when the supernatant was tipped out.

Experiment Recap and Introduction
In the previous experiment, we had managed to prove that most of the cultures of E.coli which had passed negative selection did include plasmid DNA. The only E.coli cells not to contain any plasmid were the colony 9 cells but that could be down to several reasons; not all of them down to biology.

The next step is to show that these plasmids contain the BioBricks we intended to transform the bacteria with. This can be done by using restriction enzymes followed by DNA gel electrophoresis. Each standard BioBrick is flanked with restriction sites, which are the target of restriction enzymes. The aim is to cut the plasmid at these sites; one cut leading to an open, linear plasmid and two cuts leading to the production of the BioBrick fragment along with the plasmid backbone. The two enzymes to be used today will be EcoRI and PstI

Protocol
Although there is a protocol in Dr. Aldridge's lab explaining how to do restriction digests, Prof. Wipat came up with an alternative one. The E.coli colonies selected for further processing included cultures 2, 3, 4, 5, 6 and 7. Because 6 were chosen to be further processed, 12 Eppendorf tubes were required. In the first set of six, there would be the plasmid DNA combined with EcoRI enzyme and in the second set of six tubes, there would be plasmid DNA combined with both EcoRI and PstI.

The contents to be added to the tubes containing only EcoRI include: The contents to be added to the tubes containing both EcoRI and PstI include: Of the 12 tubes, 6 of them contained plasmid DNA along with EcorI, Buffer H and SDW whereas the other 6 contained the plasmid DNA as well as EcorI, PstI and Buffer H. This was achieved using a P20 pipette. After this, the tubes were then lightly centrifuged (left to spin until 13,000 rpm was reached and then slowed down) and stored in the freezer.
 * 7 microlitres of plasmid DNA
 * 1 microlitre Buffer H (x10)
 * 1 microlitre EcoRI
 * 1 microlitre Sterile Distilled Water (SDW)
 * 7 microlitres of plasmid DNA
 * 1 microlitre Buffer H (x10)
 * 1 microlitre EcoRI
 * 1 microlitre PstI