Team:BU Wellesley Software/Notebook/TraciNotebook

Week 1: 6/6-6/10/2011

Finally iGEM kick off week arrived! I finished the BioBasics lectures with the entire team (both BU and Wellesley members were present). Following Orit's suggestion, I had the students break into teams and explain figures from a paper to the entire team to ensure students really did understand the concepts and results discussed in the paper.

Training for the wet team continued on Tuesday and Wednesday. During lab meeting on Wednesday, tasks were assigned to groups for building the first constructs the team will need to make (promoter-rbs-xFP-terminator).

These tasks included the following:

1. Choosing promoters, ribosomal binding sites and fluorescent proteins to build 4-part transcriptional units to generate fluorescence

2. Transforming the chosen BioBrick parts into Top10 competent cells

3. Miniprepping and carrying out restriction digests on these parts

4. Gel electrophoresis and extraction to confirm restriction digests

5. Ligations with the cut backbones and inserts

6. Repeat 1-5 until transcriptional units are made

Week 2: 6/13-6/17/2011

I was at SB5.0 from 6/14-6/18 and was mentoring remotely via email.

Week 3: 6/20-6/24/2011

I was away 6/22-6/25 and mentored remotely via email.

Week 4: 6/27-7/1/2011

Kyle, Alberto and I met with Jenhan and Rishi to discuss the Wet Lab needs for Batterboard. We discussed creating a different "template" for saving tubes instead of saving data in 96-well plates. We recommended creating an 81-grid box or rack in lieu of the 96-well plate since our current work has been done in 1.5mL tubes and not plates. With this 81-grid rack in mind, we want three "types" of these racks to choose from - a Plasmid rack, a Gel Extraction rack and a Frozen Stock rack to store our various types of samples.

I also worked with Swapnil one afternoon to generate and test a new liquid handling class for the robot so we can accurately pipet enzymes in glycerol.

Week 5: 7/4-7/8/2011

Given the lack of success of our transformations with ligations, I made a new batch of competent cells following the Anderson protocol using E. coli TOP10 cells. Over 200 tubes of 200uL aliquots of cells were prepared and frozen at -80C.

Week 6: 7/11-7/15/2011

I brought Vanessa and Kyle with me to talk with Jonathan Babb at MIT on Monday. We discussed our ligation problems and followed his suggestion of changing our competent cells. He gave us 5 reactions worth of their chemically competent cells to use in transformations to determine if our competent cells were the problem and not our ligations. Kyle transformed three tubes with two ligations and a positive transformation control to test the MIT cells. The two ligations yielded colonies but they were pinpoint colonies and only 1 of 8 plasmid preps grew up. Given the low success rate, it's possible that the backbones need to be dephosphorylated prior to ligation to increase our positive colonies.

While transforming with Jon's competent cells, Kyle observed that their cell density was much higher when the cells were pelleted. To determine if our cells could still be used for ligation transformations if the cells were at a higher density, Kyle, Alberto and I spun down our competent cells to concentrate them. Alberto spun down 800uLs, Kyle 1200uLs, and I spun down 1600uLs and 2000uLs to increase our cell density. We then removed enough liquid to leave 200uLs behind in the tube, resuspended the cells and then followed the transformation protocol as normal. Only one ligation transformation worked out of the various combinations, yielding no consistent result. All four attempts showed excellent results with our transformation positive control, suggesting our cells are good to use for supercoiled DNA transformation but not ligation transformation.

In order to move ahead, I purchased chemically competent cells from Bioline (alpha-select gold efficiency) for ligation transformation. I will also purchase the Zymo competent cell kit that MIT uses and generate new competent cells to try to generate our own high efficiency competent cells for future ligation transformations given the high cost of commercial cells.