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From 2011.igem.org

6/1/11-6/6/11:BU-WELLESLEY TEAM BOOT CAMP

The boot camp gave me an opportunity to meet the Wellesley students and learn more about the computational/software side of the team.

Biology:

• Reviewed a lot of terms and concepts that I had not seen in two years

• How to use restriction enzymes to cut and place different parts of the biobrick

• Saw the lab and practiced using a pipette, running a gel, etc.

• Watched an interesting background talk on tuberculosis and how we will assist in the efforts to understanding it

• TB transitions from an active to a latent state

• Hard to treat because the bacteria deceives the body's own immune system by covering itself in lipids

Computational:

• Difficulties in setting up the logic behind using inverstases

• Built a "hello world" application on Clotho as an introduction as to how create new applications

• Explored Clotho and its pros and cons

To practice techniques, we performed mini preps on a variety of previously transformed biobricks. Mine was Lac Z. The quantification of DNA isolated was as follow:

• Lacz.1 105.4 ng/microL
• Lacz.2 52.2 ng/microL
• Lacz.3 28.8 ng/microL

These sample were then run on the a gel next to a ladder and the DNA was confirmed to be present.

In the lab meeting, we began looking at the parts and the part registry page to build up some new plasmids with different parts. I worked with Shannon to find three RBS from the Anderson Collection. We chose the following based on their antibiotic resistance and strength:

1.Bba_J61101

 -On both 2010/2011 plates  (2010 Distribution Plate 1-Well 5L) 
 -Resistance A
 -11.9% (strength)

2.Bba_J61127

 -On both 2010/2011 plates (2010 Distribution Plate 1-Well 11N)
 -Resistance A
 -6.5% (strength)

3.Bba_J61100

 -On both 2010/2011 plates (2010 Distribution Plate 1-Well 5J)
 -Resistance A
 -4.75% (strength)

We then plated them and set them aside to grow.

We had the weekly lab meeting and discussed the work flow to build constructs. We also noted that our bacteria with the three different RBS had grown with two different colored colonies. We removed a piece of each colony in each sample (3 samples, 2 colonies each, 6 tubes) and did a prep for a plasmid prep.

Performed a plasmid prep on all six samples. The bacteria that grew during the prep for the plasmid prep showed stringy lifeforms and bacteria in forms that we were not expecting. DNA quantification was low, and the gel electrophoresis showed nothing notable. We then checked the bacteria samples under a light microscope with a gram stain and saw that while the Ecoli we intended to be there was there, there were also other types of bacteria that were contaminating the sample.

DNA Quantification

• Bba_J61101 A 5.2 ng/microL
• Bba_J61101 B 11.2 ng/microL
• Bba_J61100 A 2.6 ng/microL
• Bba_J61100 B 6.5 ng/microL
• Bba_J61127 A 11.4 ng/microL
• Bba_J61127 B 8.4 ng/microL

Autoclaved all our tips to make sure that they were not the source of contamination. Researched about how to build the constructs we are hoping to make.

Week of 6/12 to 6/18

At the end of the last week, we decided there were two main goals for this week. One was to figure out what was causing the contamination and preventing the transformation of the promoters and ribosome binding sites (RBS). The other was to attempt to piece together the green fluorescent protein (GFP) with the terminator. Both of these blocks had been previously transformed and were currently available to us as plasmids. We split the team up to work on these goals. Alberto and I were predominantly in charge of the first attempt of combining the GFP and terminator, which is described below. A lesser third goal was to prepare plasmids for learning about the QIA cube, a technology that would take care of such techniques as plasmid mini-preps and gel extraction for us.

• Combining GFP and Terminator

As we had the plasmids already available, our first step was to use restriction digest enzymes to cut out appropriate pieces.

GFP Gene
Size
Sites Cut 

Terminator Gene
Size
Sites Cut

We cut the plasmids at the sites noted. As the GFP gene was large enough to isolate from the plasmid, we cut it completely out of the plasmid. We only made an incision in the terminator plasmid at the EcoRI and XbalI sites in order to make room for the GFP gene. Subsequent to the restriction digest, we ran the pieces out on a gel. The result of the gel electrophoresis is the following: IMAGE The first column is the DNA ladder. The next column is the cut plasmid containing the terminator and the third one is the same plasmid without a cut. The placement of both was to be expected. As the terminator plasmid without a cut is able to supercoil, it can proceed further down the gel then the cut plasmid. The next two pieces are the cut GFP plasmid and the whole GFP plasmid. The cut one shows two pieces: the larger cut plasmid is the higher up placement. The cut GFP piece is the lower one. The GFP plasmid that is intact shows expected placement. We then proceeded to extract the DNA from the gel. The blocks we desired to remove were the GFP gene and the terminator plasmid. After running the gel extraction protocol on these blocks we used the Nano-Drop to quantify how much DNA we removed. GFP ng/microL Terminator ng/microL We were hoping for large amounts, but proceed onto ligating these pieces together anyways. After running the ligation protocol on the two pieces, we proceeded to use the new, combined plasmid in transformation. The goal of this was to acquire Ecoli who would pick up the plasmid.

QIA Cube

RBS and Promoters