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# Notebook

Preparing/Autoclaving A media - 6/10/2014

**A media:**

Made 750 mL of A media

Weighed:

9g Pancreatic Digest, 9 g Difco Yeast Extract, 3 g Dextrose, 3 g KH2PO4 monobasic, 0.75 g MgSO4 \* H2O, 750 mL distilled water

Poured into a plastic beaker and stirred for 40 mins.

Then placed in refrigerator at 4°C for 40 mins.

Poured 75 mL aliquots of media into 200 mL bottles.

**For 200 mL storage bottle (Before autoclave):**

1. Put water in the container that will hold all the aliquoted bottles
2. Put foil on lid of aliquoted bottles
3. Open the lids of the bottles slightly but not completely
4. Put autoclave tape on the foil

**To autoclave:**

1. Use heat-resistant gloves to place container that contains bottles in autoclave
2. Close autoclave with foot pedal
3. Set up a username and password
4. Choose liq60 = 1hr. and 32 mins.
5. Wait for loud sound which means that the autoclave is compressed
6. Take back cart and write down what time you started autoclave

**After autoclave:**

1. Put on normal and heat resistant gloves
2. Slide the container with bottles onto cart
3. With heat gloves on, close the lid of each bottle and place outside container
4. Get rid of water in container
5. Place bottles back in container
6. Wait for bottles to reach room temperature
7. PLACE LABEL ON EACH BOTTLE WITH: NAME OF MEDIA, “AUTOCLAVED”, DATE, and NAME or INITIALS.

Culturing P. Acnes – Day 1 – 6/17/2013

**For cultures at 24 °C:**

Started with 15 mL of P. Acnes in enriched clostridial broth.

Pipetted eight 1mL aliquots of the liquid culture into eight 125 mL glass bottles near a flame.

Tightly closed lids of bottles.

Then individually opened autoclaved A media and aliquoted glass bottles of P. Acnes liquid culture near flame.

Poured approximately 30 mL of A media into each glass bottle.

Tightly closed bottle.

Put in autoclaved stirbar wrapped in aluminium foil into bottle without touching the stirbar.

Then placed bottle in 1.3 L LockandLock container.

Placed anaerobic detector in chamber.

Loosened the lid of glass bottle, placed anaerobic Gaspak in chamber and quickly closed lid.

Placed bottle on Corning stirplate at value of 4.

This was repeated with 3 other glass bottles.

**For cultures at 37 °C:**

Started with 15 mL of P. Acnes in enriched clostridial broth.

Pipetted eight 1mL aliquots of the liquid culture into eight 125 mL glass bottles near a flame.

Tightly closed lids of bottles.

Then individually opened autoclaved A media and aliquoted glass bottles of P. Acnes liquid culture near flame.

Poured approximately 30 mL of A media into each glass bottle.

Tightly closed bottle.

Placed two bottles in each 1.3 L LockandLock container.

Placed anaerobic detector in chamber.

Loosened the lid of glass bottle, placed anaerobic Gaspak in chamber and quickly closed lid.

Then placed container on a shaker in a 37 °C room.

There were two 125 mL bottles in each chamber.

**Stored excess liquid culture in a centrifuge tube at room temp.**

**Created solution of ½ mL of P. Acnes liquid culture and ½ mL glycerol in microcentrifuge tube and placed in -80 °C freezer.**

**Possible errors:** We did not use pipette tips that had been autoclaved L for transferring 1 mL of P. Acnes liquid culture into glass bottles.

6/18/14 – Plates for P. Acnes ATCC\_11827 culturing

7 out of the 8 cultures are still pretty clear. All the anaerobic indicators in the chamber were still pink which means that the chambers are still anaerobic and the LockandLock is working well.

**Possible source of error:** One of the glass bottles in the 37°C rooms anaerobic chambers is much cloudier than the others. Carlos said this is fine and is most likely the result of us getting a clump of bacteria when we added it to the media.

We made 100 mL of A media for plates with this adjusted recipe:

|  |  |
| --- | --- |
| **Component** | **100 ml** |
| Pancreatic Digest of Casein A | 1.2 |
| Difco Yeast Extract | 1.2 |
| D+ Glucose (Dextrose) | 0.4 |
| KH2PO4 monobasic | 0.4 |
| MgSO4\*7H2O | 0.1 |
| Difco Agar | 1.5 |

dH2O 100 mL

Firstly, we mixed everything except the agar in an Erlenmeyer flask covered with aluminium foil. This was stirred on a stirplate for 40 minutes. We then placed it in a -4°C refrigerator for 32 minutes. After taking it out of the fridge, we put in our agar. The flask was then placed in a bin with water and autoclaved for 1hr. and 2 minutes.

**Using water bath:**

1. Press set and wait
2. Then while holding down set, increase temperature

After the A media came out of the autoclave, we tried to place it in the water bath at 55°C for ½ hour, however the flask floated in the water bath so we let it cool at room temperature. We let it cool for too long so it was no longer completely homogenous. We put it in the microwave for a period of 10 seconds four times.

**Procedure for creating plates:**

1. Put on gloves
2. Wipe down space with ethanol
3. Turn on fisher burner
4. Place plates around the burner
5. Spray some ethanol onto a tissue and only handle autoclaved flask with this tissue

1. Take foil off autoclaved flask with media which should be at 55°C or cool enough to hold without burning your handv
2. Flame top of the flask for a couple of seconds
3. Open lid of petri dish individually and pour on media until petri dish is ½ to 2/3 full
4. Then close lid most of the way but leave a little bit open
5. Repeat this for the other three petri dishes
6. Let the petri dish cool for at least half an hour or until the condensation on the lid has completely disappeared.
7. ENSURE THAT THE MEDIA IS SOLID
8. Then close lid and seal the petri dish with a strip of Parafilm.
9. Label the plate with date it was created, the media used, and name.

**Possible source of error:** We had a stir bar in the flask which we autoclaved. This fell into one of the petri dishes. We used two scoopulas, which were flamed with ethanol, to take the stirbar out of the petri dish. We also then flamed the media in the petri dish using the fisher burner. This petri dish was marked with an X.

6/19/2014 – ATCC\_11827 strain cultured

We put in 1 mL of reinforced clostridial media into the ampule of P. Acnes, which we received from ATCC, using a syringe. Then, 700 µL of the resuspended P. Acnes was syringed into a microcentrifuge tube. The tube was opened next to the fire and 200 µL of the bacteria was pipetted into a 250 mL glass bottle. We also poured approximately 20 mL of A media into the glass bottle. This bottle was placed in a LockandLock with a Gaspak.

**Liquid Culture**

We put in 1 mL of reinforced clostridial media into the ampule of P. Acnes, which we received from ATCC, using a syringe. Then, 700 µL of the resuspended P. Acnes was syringed into a microcentrifuge tube. The tube was opened next to the fire and 200 µL of the bacteria was pipetted into a 250 mL glass bottle. We also poured approximately 20 mL of A media into the glass bottle. This bottle was placed in a LockandLock with a Gaspak.

**Plates**

1. Put on gloves, wipe table with ethanol, turn on flame.
2. If using plate spreader, dip in 90% ethanol and flame. If using loop, heat platinum loop until red hot and let cool for 30 secs – 1 min.
3. If using platinum loop, after cooling touch onto gel and make sure it is cool and does not melt plate.
4. If using plate spreader, open plate next to flame and pipette 50 µL into the center of the plate. Then spread around the plate using a mini rotating bench.
5. If using platinum loop, then dip loop in suspended bacteria and streak on plate.
6. Close petri dish when not streaking.

**We made four plates.**

The first plate was done with a plate spreader using 50 µL of bacteria. This was placed in a LockandLock with a Gaspak in the 37°C room on the shaker.

The other three plates were streaked. Two of these plates were stored in a LockandLock under anaerobic conditions in the 37°C room on the shaker.

The last plate was stored under aerobic conditions in the 37°C room on the shaker as a control.

**Leftover ATCC\_11827**

We still had 450 µL of ATCC\_11827 left in the microcentrifuge tube. This was mixed with 450 µL of 50% glycerol and stored in a -80°C freezer.

6/20/2014 – Got dam- E. Coli strain

We got the dam- E. Coli strain and placed in the -80°C freezer

Two of the glass bottles in the 37°C room were very cloudy and the rest were still somewhat cleara

In the 24°C room (lab), the cultures definitely have growth but not as cloudy as in the 37°C room.

6/23/2014 – Got dam- E. Coli strain

**Plates with Erythromycin:**

|  |  |
| --- | --- |
| Component | 200 ml |
| Pancreatic Digest of Casein A | 2.4 |
| 2.4 |
| D+ Glucose (Dextrose) | 0.8 |
| KH2PO4 monobasic | 0.8 |
| MgSO4\*7H2O | 0.2 |
| Difco Agar | 3.0 |
| dH2O | 200 mL |

Made a 50mg/ml stock solution of erythromycin in ethanol

After autoclaving the plate media put in 200 microlitres for every 1ml

So if making 200 ml then 40 microlitres of erythromycin

Username: grab

Pwd: 6428

**Phenol: Chloroform Extraction**

We used the protocol Carlos gave us.

**O/D for most turbid strains**

Most turbid strain had an O/D of 1.6. We decided that this would be used for further P. acnes strain 6919 cultures because it was too saturated.

The O/D of the second most turbid strain was 0.556. This was a good O/D for adding glycine. We added 0.75 mL of 20% glycine into our 30 mL culture as a cell wall weakening agent.

We also now have a stock solution of E. Coli transformed with the ampicillin resistance. In addition, we have a glycerol stock of the transformed DNA.

6/24/2014

Took the O/D of two of the cultures in the 37°C room. One was 0.354, the other was 0.202. Not ready for glycine yet. The cultures in the lab are not ready for glycine yet. We also got lysozyme and figured out how to get the glycerol.

6/25/2014

Took the O/D of two of the cultures in the 37°C room. One was 0.707, the other was 0.262. We added 0.50% glycine (0.75mL). The other culture was not ready for glycine. We also checked the O/D of the culture in the 37°C room which was previously at 0.556. It had actually decreased to 0.515.

We got the DNA back from the Phenol:Chloroform Extraction. We have to run it on a gel to determine if there is a lot of RNA present.

Made the Cheong buffer but did not autoclave. The recipe was for 272 mM sucrose + 7 mM sodium phosphate + 1 mM MgCl2. We decided to make 500 mL of the Cheong Buffer. This meant we added 46.552 g sucrose, 0.49686 g sodium phosphate, and 0.0476 g MgCl2.

6/26/2014

We made a 5 mL liquid culture of transformed E. Coli. We added 5 microliters of ampicillin to the liquid culture. This was left in a refrigerator for 14 hours overnight.

We added 20 microliters of TE buffer to the plasmid that Carlos gave us from the P/E extraction because there was too little of it present.

We nanodropped this sample and got a concentration of 0.2946 ng/microliter.

**Making the gel**

Need 25mL of media for the gel.

Should be 1X TE buffer

Concentration of agarose is 0.8 g/100 mL

Pour all of it into the flask and put towel stopper on flask. Microwave for around 1 minute in order to melt all the agarose.

Add ½ microliter of ethidium bormide (in red microcentrifuge tube)

Make sure comb is in. Pour into well.

**Running the gel**

Once the gel had set we added around 250 mL of TX buffer. The wells are connected to the black cord (negative). We added 1 microliter of the ladder in the first well, 5 microliters of pNIT linear plasmid + 1.25 microliters of loading dye in the second well, and 5 microliters of pBres36a plasmid + 1.25 microliters of loading dye in the third well. This was run at 110 kV for 1.5 hours.

**Result of gel**

When we checked the results of the gel, we were able to determine that the size of the pBres36a plasmid was 8 kb and that there was no RNA present as a result of phenol:chloroform extraction. However, the pNIT linear plasmid was not visible on the gel.

Took the O/D of the culture in the 37°C room which was 0.707. It was now 1.065, which means that it is ready for electroporation. We did an electroporation protocol (std 6/run13) for this culture. However, we did not add plasmid yet because we did not have enough. The cells were stored in the -80 room.

6/27/2014

We made TGE buffer for electroporation.

6/30/2014

We made to more cultures of ATCC\_6919. We added 1 mL of the overgrown solution to 35 mL of A media. The O/D of the stock culture was 1.686. This was placed in the 37°C room

7/1/2014

We did a two phenol:chloform extractions yesterday. One was with E. Coli grown in A media. The other was with E. Coli grown in LB media. We thought we didn’t have success with the extraction with the E. Coli grown in A media. We did get DNA in the extraction, with the E. Coli grown in A media.

7/2/2014

We did three phenol:chloroform extractions with E. Coli grown in A media.

We also checked the O/D of two cultures of ATCC\_6919. One was at 0.737 the other was at an O/D of 0.765. We ran 14/9 (std/run) and 10/2 on the two respectively. This meant we added 1.5 mL of glycine to both.

7/3/2014

Diluted 30 µL of DNA with 20 µL of TE 1X buffer.

DNA Sample #1 – 2 µg/µl

DNA Sample #2 – 3.2 µg/µl

DNA Sample #3 – 4.5 µg/µl

O/D’s of the two cultures today - 0.997, 0.971

DNA Sample #3 – 3.0 µg/µl – 80 µL total

DNA Sample #1 – 0.76 µg/µl – 160 µl total

We conducted two electroporations today. Std/run 10/2 and Std/run 14/9. We used DNA sample 1. Both arced. We suspect it might be because of the Cheong buffer, which contains magnesium chloride (a salt).

7/7/2014

We conducted four electroporations today. Std/run 10/2, Std/run 14/9 (twice), and Std/run 2/1.We used DNA sample 1. Std/run 10/2 and Std/run 14/9 (twice) both arced but Std/run 2/1 did not. So we could conclude that the Cheong buffer was the reason for the arcing.

We also made 6 A media plates without erythromycin.

7/8/2014

We made A media

7/9/2014

Did electroporation on 1/8

Started new liquid cultures:

2 11827 upstairs

1 6919 upstairs

3 11827 downstairs

1 6919

7/10/2014

Plated 1/8.

7/11/2014

Checked the OD of all of the liquid cultures in 37 C. 3 of them had a high enough density to add glycine. They were roughly 0.56, 0.68, and 0.82. I assigned trials to each of these three (6/13 for the ATCC\_6919 and 12/15 and 8/12 for the two ATCC\_11827). The fourth culture I checked had an OD of roughly 0.4, so I was hoping to add glycine on Monday.

7/14/2014

I was not able to get into the building over the weekend, so two of the four cultures had overgrown in that time. The OD of both of these were over 1.4. The two that remained were both ATCC\_11827 that I had added glycine into, so I made competent cells from these and tried to perform an electroporation. The OD of 8/12 was approximately 1.05 and the OD of 12/15 was approximately 1.2. The machine sparked, but it did so between the machine’s metal and the metal on the electroporation cuvette. Does this constitute an arc? I took it to be an arc at the time, and so disposed of the cells and the cuvettes, but this may have been an error in retrospect. I saved several microcentrifuge tubes of each trial in the -80 freezer for later use.

We also discovered at the team meeting that Cheong had not actually GROWN the bacteria at different temperatures, but incubated them at that temperature for 5 hours after growing them, but before making competent cells from them. I may start trying this technique if the previous technique did not work.

Also, glancing at the plates for our previous electroporations, I could see that the plate with no erythromycin showed a lot of growth on it, with not much on the erythromycin plate. This shows that the cells were not killed during the competent cell preparation or the electroporation. I will look in more detail in the next few days to check for small colonies.

7/15/2014

Today I started 2 new liquid cultures in the 37 C room, and updated the lab notebook. I may perform electroporations from the frozen competent cells a little later, but no promises.

We only have 2 plates with erythromycin on them remaining, while 4 plates with no antibiotic are still available for use. Some of the erythromycin plates somehow got contaminated with bacteria, so they had to be thrown out. I will need to make some more of each of these plates in the next few days.

7/16/2014

Today I made 4 plates with erythromycin and checked the plates downstairs for growth. There did not seem to be any growth on the plates with erythromycin on them, but there was growth on all the plates without this resistance. This indicates that I did the experiment correctly (or at least mostly) and that the cells didn’t die during the procedure, but there were no transformants. Ching came to look with me, and said also that he didn’t see any transformed colonies.

I made a stock solution of approximately 1 mL of 50 mg/mL erythromycin in ethanol, then added 20 uL of this into a 100 mL batch of A media with agar. I also saved 100 mL of A media with no agar and no erythromycin in a bottle for later use. The four plates look fine and are now in the fridge, ready for use.

7/17/2014

Today, I checked the OD of the cultures I started on Tuesday. The were both approximately 1.2, much too high to add glycine, so I threw them out. These were both of strain ATCC\_11827, and grew much faster than I would have expected.

We also discovered that Cheong’s protocol was actually slightly different from ours in that he grew p. acnes to stationary phase only at 37 degrees C, then aliquotted it (100 uL) into several 5 mL tubes of growth media. They then incubated it for 5 hours at multiple temperatures (for it to get into exponential phase, presumably), then made competent cells.

Carlos (who got back today) told me that this is essentially what we have been doing, unfortunately after I had already dumped the cultures we had growing upstairs because I was confused about the implications of this difference.

Ching suggested a few ideas to optimize our protocol. These ideas were: plating ALL of the cells after recovery instead of just a portion of them (do this by spinning down the cells, taking out most of the media, then resuspending the cells in less media and plating them. He also suggested varying the temperature of recovery, because a lower recovery temperature may inhibit the restriction enzymes in p. acnes.

7/18/2014

Today, I made 2 liquid cultures of p. acnes to grow in 24 C, because it will take longer, and so the weekend will give these cultures a head start. I also emptied all the anaerobic (Lock-n-Lock) chambers that I could and cleaned bottles that I needed to use for next week.

Stephen and I also discussed the project, and how to optimize our protocol, because we decided that the Cheong buffer was most likely not beneficial to the project (it contains salts, which leads to arcing). We changed this parameter to the incubation temperature of the cells during recovery.

Today, I did notice a single colony on one of the plates with erythromycin, which Carlos said could probably be p. acnes, although it would need to be verified. Even if it is p. acnes, he said, one transformation isn’t a high enough efficiency to actually mean anything.

7/21/2014

The ATCC\_11827 strain miraculously grew too much over the weekend while the other strain did not. I suspected not only p. acnes was present in the culture, so Carlos showed me how to check this by using the phage, and gave me some phage so I could check for this in the future. I am currently waiting for it to grow up (overnight) to see if there was contamination. This was the same strain that grew too quickly last week, so I have reasons for my suspicions.

I also discovered that one of our bottles of media had been contaminated, so I disposed of it and will probably have to make some more later (there are only 160 mL remaining).

I started 2 new liquid cultures of ATCC\_6919 to incubate in 37 C. Hopefully they will grow quickly. I also measured the OD of the not-overgrown ATCC\_11827 culture. It was 0.155, so too early to add glycine.

7/22/2014

I added glycine to the ATCC\_6919 that was growing upstairs, because it was definitely ready, just by sight (hopefully it is p. acnes). It will be run 11/16 on the new chart (version 2), and I plan to make competent cells from it tomorrow. I also checked the plate downstairs with the phage added into it and there was definitely another type of bacteria than just p. acnes growing, and the phage hadn’t disposed of it. Today, we are going to do the same experiment for the overgrown culture of ATCC\_11827.

Today I also made more media (3 bottles of 60 mL without agar, and 1 bottle of 120 mL with agar).

7/23/2014

Today I made competent cells from the 11/16 culture. The OD was 0.938. I will also try an electroporation with these comp. cells and will plate them tomorrow using the modifications. The parameters are: 0.6 lysozyme (which I made this morning), 2.00 ug DNA, 15 kV/cm electric field, 37 C post-zap temperature, and no TypeOne present.

I added glycine into the 2 cultures that were growing in 37 C. The two OD’s were approximately 0.51 and 0.42. Tomorrow or the next day, I plan to make comp. cells from these. They will be runs 15/3 and 3/11, respectively.

7/24/2014

Plated the electroporated cells from yesterday.

I also checked the OD of the two other cultures, which came out to be 0.819 and 0.816. I then made comp cells from these two cultures, which (again) are runs 15/3 and 3/11. I then electroporated one round of these comp. cells.

We also discussed how to check if the colony that was on the previous plate (run 1/8 from the old list) was actually p. acnes and if it was actually transformed. I will need to grow it up on a plate with erythromycin (or in a liquid culture) anaerobically, then streak it aerobically to make sure it’s p. acnes, and perform some PCR to verify that the vector is in there.

7/25/2014

Plated the electroporated cells from yesterday.

Discussed with Carlos and Ching how to check the transformant, and started a small liquid culture (in erythromycin) as well as plating the cells on an erythromycin plate.

I then created two new liquid cultures of ATCC\_6919 to incubate in 24 degrees.

7/28/2014

First thing in the morning, I checked all the plates and liquid cultures. There seemed to be some colonies on the run 15/3 plate, but the others I couldn’t see very well. The liquid culture with erythromycin in it (to check for the transformant) was definitely grown up, maybe too much so I suspect it has something to do with how it was kept, although it is also possible that the erythromycin didn’t make it all the way into the media. One of the two liquid cultures in 24 C seemed to have grown up enough to check the OD, so I’ll do that today, and will likely add glycine to it. Finally, the plate with the phage treatment on it was still not entirely grown up in my opinion, which is a bit strange. I’ll ask Carlos what he thinks.

The OD of the liquid culture was below 0.4, so I have decided to give it another day. Also, Carlos (or that entire side of the Hatfull Lab) is not here yet, so I will have to hold off on asking any questions.

Carlos arrived and checked the plate, saying that it is p. acnes because a section of the lawn was missing, right where the p. acnes phage was placed. I can now make cultures from strain ATCC\_11827.

07/29/2014

Today, the OD of the cultures I started last Friday were 0.513 and 0.462, and so are ready to have glycine added to them. These will be trials 7/6 and 13/5, both of which have 1% glycine added into them. Also, these are the last competent cells I will have to make of strain ATCC\_6919.

I plan to pour more plates today (with erythromycin added into them) from the agar that I saved from before. I will be adding 24 uL of erythromycin to the entire batch before pouring. The media hadn’t completely melted (or had resolidified), so much of it was wasted on a bad plate before 4 more plates were made correctly. Thus, I now have 7 plates with erythromycin on them with which to complete this project for the summer.

These are the competent cells I have in the -80:

(from old list): 2/1, 8/12, 1/8, 12/15, 14/9, 10/2

which translate to (on new list): 9/10, 5/9, 1/2, 15/3, 4/5, 6/13

Cells in -80 (new list):

11/16, 3/11, 15/3

07/30/2014

I checked the OD’s of the two cultures with glycine added. Trial 7/6 was 0.898 and trial 13/5 was 0.573. I decided to make comp. cells from trial 7/6, but not from 13/15, which I will leave until (probably) Friday.

I should also mention a change that I have been making to the protocol. The high level for electric field is now 12.5 volts/cm, because this is the highest that their electroporator can go with 2 mm cuvettes. I have used this level for all of the new trials.

07/31/2014

Today was Science Palooza, but I still was able to add glycine to the two cultures growing in 37 degrees. Their OD’s were 0.792 and 0.896, so I let them sit off of the shaker overnight as they grew with glycine.

I also checked the plate that corresponded to run 11/16, and the plate somehow contained a lawn. The other two trials will be officially checked tomorrow, but I can already see a decent amount of transformed colonies.

08/01/2014

Today, I made competent cells from all three cultures that had been growing with glycine (runs 10/8, 13/15, and 14/16), and saved them in the -80. This was after Stephen and I decided on a plan to move forward, which includes verifying the colonies on the plates we already have with transformed colonies, and then waiting to do the rest of the electroporations until the semester starts. I now have 9 different preparations of competent cells in the -80 that could be readily electroporated.

I discussed with Carlos how to check the “transformed colonies” that we have, and he explained that I would need to streak each of the colonies (multiple on one plate), and start a small liquid culture of one of the small colonies, and one of the big ones. He gave me the code to get in and do all of this tomorrow, before waiting all of next week. He also showed me how to easily count the colonies using their lab equipment, so yay. For the plate with the lawn, I need to restreak it on erythromycin plates and hope for single colonies.

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