Week 2

7/1/13
We prepared antibiotic stocks that would be used in our Luria broth media. After adding the appropriate ingredients and autoclaving the mixture LB media, we added different types of antibiotics that would later be used in screening for transformations the following day and poured plates. We also hydrated the following parts and plasmids of interest:

• B0015
• J23101
• B0034
• E0040
• pSB1A3
• pSB1K3

The parts were then stored in the freezer at -20⁰ C.

7/2/13
Each of the parts that were hydrated on Monday were then transformed into competent cells through heat shock. After carrying out seven different transformations, we emailed Dr. Yokobayashi about meeting to discuss our project with regards to riboswitches. We also spoke to one of our advisers about using TAL repressors in our construct and possible methods of assembly. We also began creating and updating different parts of our Wiki including the Notebook and Safety Overview section.

7/3/13
We had a meeting with Dr. Yokobayashi about using different riboswitches. He asserted that the use of different ribosomal binding sites may inhibit the function of the riboswitch. Instead, we should plan on altering the sequences upstream of the Shine-Dalgarno sequence in an attempt to establish variability in function. We decided to start by using a theophylline riboswitch from the literature. After the meeting, we looked at our transformation plates from the day before and only our positive control seemed to work. To mitigate this setback, we revived some cultures in our glycerol stock and did an additional transformation from the 2012 distribution kit. We also made new cultures containing three different TAL repressors with different activities. While waiting for certain experiments to finish, we continued improving different parts of our Wiki.

7/4/13
In celebration of 4th of July, we took the day off and decided to come into lab on Friday. One of our advisers was kind enough to check on our bacteria cultures from the day before.

7/5/13
Today, we performed DNA extractions for our three TAL repressors, J23101, B0015, and E0040. The cultures containing the three different TAL repressors were also stored in glycerol stocks. In addition, we practiced using Golden Gate assembly on several parts involved in an oscillator construct. We calculated the correct concentrations for each individual part of the construct (mRFP, terminator, RBS, and araC promoter) for correct assembly. In the past, we used 10 cycles to create the correct construct of interest, but we decided to do 50 cycles instead to ensure success in assembly. Another part found in the registry BBa_K598001 will be used as our riboswitch reporter to ensure that the theophylline riboswitch is functioning. The part was hydrated and used in a transformation. With consideration of several other unsuccessful transformations, we decided to use additional DNA (2 uL instead of 0.5 uL).