Team:Calgary/9 July 2009

• Today I ran a colony PCR of my J13002-LuxOD47E-B0015 colonies to verify the presence of the B0015 terminator in my construct. BBK-CP forward and reverse primers were used with p Taq and cycling conditions were as follows: 94°C for 6 min, 36x(94°C for 30 s, 55°C for 45s, 72°C for 90s), 72°Cfor 10 min, held at 4°C. PCR products were visualized on a 1% agarose gel run at 120 V with J13002-LuxOD47E and LuxOD47E as size controls. Gel is pictures below. Lanes 1-4 are trial one where the B0015 terminator was treated as the insert. Lanes 5-8 are trial 2 where the J13002-LuxOD47E construct was treated as the insert. Lane 9 is J13002-LuxOD47E and Lane 10 is LuxOD47E. Lane 11 is blank and Lane 12 is the negative control.

• Because all bands in the first 9 lanes appear to be the same size as the band in lane 9 with J13002-LuxOD47E, we can conclude that the B0015 terminator was not successfully cloned in and we will have to go back to contruction again.

• Performed a restriction digest with EcoRI, XbaI and SpeI of B0015 and the J13002-LuxOD47E construct, digesting the insert with EcoRI and SpeI and digesting the recipient with EcoRI and XbaI. Left the digestioin in the 37C waterbath for 2 hours, transformed into TOP10 cells on both AC and C plates and left for overnight growth.

1. Single construction site for ligating two parts of the circuit together. These two parts must have been cut with the proper restriction enzymes to place one before the other ie. Promoter cannot be placed behind RBS.

2. Multiple construction site that works the same as the first but with two parts already together that must be connected to another part.

3. Multiple construction site with three pieces together already. There is also an option to ligate Promoter/RBS with Gene/Terminator, which can actually be considered a shortcut as long as users understand what they are doing.

One flaw in the activity is that you are only able to cut each piece once in the donor and recipient tubes, but I thought it would be unnecessarily tedious to cut at each step of construction once they have already succeeded in doing it once, but this can always be changed although I do not plan to in the upcoming week. It would involve possibly doubling the amount of comparisons within the donor and recipient tubes avatars may use to determine what they wish to cut. I also began to modify the recipient and donor tube scripts in order for them to cooperate with the three construction sites so they are no longer fully functional as I continue to change them. I have determined the names of the products an avatar may obtain from performing the activity so now I will have to create them and request they be given to inventory, which I hope to complete by next week and will be starting tomorrow.

I will most likely finish the restriction digest notecard in parallel with its corresponding activity and I will be sending these notecards to Mandy once again to look over and examine their validity, which I plan to have done by Monday (July 13). I was also able to construct the basic plates for streaking for the bacterial transformation activity.

I was also able to complete the script for the sequencing station as per Mandy’s request and it will now give any avatar who drops ‘Amplified Colison E2’ into the sequencing object a notecard, which as of now does not contain any useful information. I will be adding various other amplified DNA products as objects to give back sequencing for on Friday, which may continue into next week.

From it, one can clearly see that RFP is glowing much brighter than the negative control, and that GFP is, although less obvious, also glowing. No other lab work was performed on this day.