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From 2011.igem.org

• Protocols

Contents 1 Week 1: 6/1-6/10 2 Week 2: 6/11-6/17 3 Week 3: 6/18-6/24 4 Week 4: 6/25-7/1 5 Week 5: 7/2-7/8

Week 1: 6/1-6/10

The first few days, the group sat through a boot camp, where we learned about biobricks, the basics of Biology such as DNA transcription and translation, and how to use Clotho tools. As exposure to the more computer science aspect of the project, the group learned how to write a Clotho application.

As a test run, mini preps (protocol link) were performed on a few samples of biobricks that were previously plated and transformed. Most of these samples unfortunately did not yield a high concentration of DNA, except for a few. After quantifying the samples, the samples were loaded on a gel next to a ladder to confirm the DNA that was present. However, most bands did not appear on the gel after exposed to the UV light. This concluded that there was no DNA in the samples that were being looked at. Next, several transformations were made for biobrick parts such as RBS, RFP, and constituent and inducible promoters.

The transformations produced several cultures that were both dark and light in color. Plasmid preps were made of the dark and light cultures of the UV promoter. The dark culture had an okay amount of DNA concentration. However the other samples did not have a good concentration of DNA either.

There seemed to be contamination or something else growing on the plates because some of the cultures were oddly shaped and there was a pungent smell. As a result, a sample of plasmid prep culture was stained and looked under the microscope. Most samples did not have E. coli in the sample.

Week 2: 6/11-6/17

A second round of plasmid preps were made of the same plates to troubleshoot the cultures that do not have high concentrations of DNA. These unfortunately still did not work or produce anything. Only the UV promoter sample had a culture.

This week the BU Wellesley team is demoing the QIAcube from Quiagen. Plasmid prep of the UV sample was used to start up and learn how to use the QIAcube.

It seems that the problem is the plates with ampicillin. A sub-group of our team were working on putting together the GFP (Bba_J52028) and terminator (backbone, Bba_B0015) to test out various RBS and promoters combinations. Once the GFP and terminator were ligated together, the construct was transformed on a Kanamycin plate. This successfully grew on the plate. Other parts were transformed and grew nicely on the Kanamycin plates. As a result, it is believed that the ampicilling plates are not selecting because there was also a negative control plated with just top 10 cells and there was some growth seen on the plates.

New ampicillin plates were made.

New transformations were made of the 7 original parts chosen and few additional ones (RBS, promoters, RFP). Most of these seem to grow successfully, however 2 plates had cultures with a pink tint to them. There was one plate where nothing grew on it. There was also two negative controls for ampicillin plates and kanamycin plates, where nothing grew. Plasmid preps of the new transformations were made.

Wells 1 through 9 and M being the location of the ladder. 
1. Bba_I13453  18.6 ng/uL
2. Bba_J23100  53   ng/uL
3. Bba_J61127  23.1 ng/uL
4. Bba_E1010   56.5 ng/uL
5. Bba_J23101  32.6 ng/uL
6. Bba_14053   17.5 ng/uL
7. Bba_J23109  31.6 ng/uL
8. Bba_J61101  9.6  ng/uL
9. Bba_J61100  30.2 ng/uL

Week 3: 6/18-6/24

- Prepared plasmid preps and continued to used the QIAcube for minipreps of biobrick parts listed below:

Composite Parts:

 -  Bba_E0430 (YFP composite) 1/A/8I
 -  Bba_E0240 (GFP composite) 1/A/12M
 -  Bba_R2000 (Promoter) 1/K/16F

Reporters:

 -  Bba_E0020 (ECFP) 1/A/6A 
 -  Bba_E0032 (EYFP) 1/A/6E
 -  Bba_K156010 (BFP) 2/A/4D

- Restriction digest on Bba_E0430 (yfpc), Bba_E0240 (gfpc), Bba_R2000 (promoter), Bba_J61127 (RBS), Bba_J61100(RBS), GFP+Term(2)

- More plasmid preps of promoter and RBS from 6/15 transformations.

- Prepare for joint meeting with Professor Densmore

- Gel electrophoresis with restriction digested parts and gel extraction with the QIAcube. Quantification values of the gel extraction were somewhat low.

- A restriction digest was performed on BFP and RFP to make up the list of reporter genes that would be ligated with a terminator in route to make one device.

- The gel ran once more with digested parts because the bands on the gel looked very faint and did not seem to produce the correct bands according to the parts being cut.

Wet Lab Meeting:

Two approaches were decided on to make the 4 different successful devices, the Bottom Up approach and using composite parts with flourescent proteins to build the some of the devices. The team broke up into two. I became part of the team, where we use the composite parts approach using YFP, GFP, and RFP. I am responsible for making a device with the yellow fluorescent protein composite parts along with four different promoters found in biobrick parts. The promoters include Bba_I13453 (pbad), Bba_I14033 (pcat), Bba_R0040 (ptet), and Bba_R2000.

To Prepare for Ligation of device:

- Transformation of Bba_R0040 (ptet)

- Restriction digest, gel run and extraction with Bba_I13453 (pbad), Bba_I14033 (pcat), and Bba_E0340 (YFPc) - Restriction Digest: Bba_I13453 cut with SpeI and PstI

Ligation:

 Five samples were made at 3:1 ratio of insert to backbone. 
    -  pcat + YFPc
    -  pcat + YFPc
    -  pbad + YFPc
    -  pbad + YFPc
    -  pbad + YFPc

Transformations of these samples were done, but unfortunately nothing grew on the plates.

Week 4: 6/25-7/1

- Restriction Digest 3 samples of YFPc with Xbal and PstI and R2000 with SpeI and PstI.

1. Restriction digest of Bba_E0430.1 (YFPc)
2. "        "        "         "
3. Uncut Bba_E0430.1 (YFPc)
4. Restriction digest of Bba_E0430.2 (YFPc)
5. "        "      "           "
6. Uncut Bba_E0430.2 (YFPc)
7. Restriction digest of Bba_E0430.2 (YFPc)
8. "      "            "         "
9. Restriction digest of Bba_E0430 (YFPc)
10. "        "       "        "
11. Restriction digest of Bba_R2000

- These samples ran on a gel, extracted, and quantified. Gel quantification values were ranging in the 20s for the samples listed above.

- Restriction Digest of Bba_I14033 (pcat), Bba_I13453 (pbad), Bba_R2000, Bba_E0240 (GFPc), and Bba_R0040.

- A two 24 well gel was made with 2 wells per each sample. 10 wells were filled with samples listed above.

- These samples were extracted and quantified. The concentration values of the samples were all in single digit.

- Ligation:

 Five samples were ligated at a 6:1 ratio of insert to backbone. 
   -  Bba_R2000 + Bba_E0430
   -  Bba_I14033 + Bba_E0430
   -  Bba_R0040 + E0430
   -  Bba_R2000.1.2 + E0430
   -  Bba_I13453 + E0430 

. These 5 ligation samples were also transformed, but nothing grew after incubating them overnight at 37 degrees Celsius.

A new ligation protocol was attempted for two new samples. The ligation samples are Bba_R2000 + Bba_E0430 with one sample being a 3:1 insert to backbone and the other 6:1. The samples will be held at 16 degrees Celsius for 12 hours and holding it at 4 degrees Celsius for 4 hours.

Week 5: 7/2-7/8

The new ligation samples were transformed over the weekend on plates. On Tuesday morning, there was growth on the plates; however, there weren't any colonies growing, just a lawn of bacteria. It was concluded that the plates may have not had the antibiotic to be able to grow the specific parts that were ligated. Another attempt was made, but as plasmid preps were made it was discovered the LB broth had some contamination. This added to the transformation problem because the same LB broth was used to plate the samples. Because of contamination, new LB broth was made as well as new plates with the appropriate antibiotics.

Another attempt to transform last week's ligation samples, but unfortunately again no growth.

While the ligation issues are being figured out, stocks of parts that are needed for future ligations are being made.

Three promoters needed in gel extraction stage are promoters, Bba_I13453, Bba_R0040, and Bba_I14033. These biobrick parts were cut with SpeI and PstI using a higher mass of DNA.

These pieces were run on a gel providing some strange unclear results on the image below.

Wells:

5. Restriction Digest of Bba_I13453.2 
6. Uncut Bba_I13453.2
7. Restriction Digest of Bba_R0040
8. Uncut Bba_R0040
9. Restriction Digest of Bba_I14033
10.Uncut Bba_I14033

- Only the well 5 and 7 were cut and quantified.

- Plasmid preps of various parts: Bba_E0430, Bba_I14033, Bba_I13453, Bba_E0240

- Streaked 3 plates from -20 degrees Celsius stock (Bba_R2000, Bba_R0040, and Bba_I13453)