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WEEK 1: 6/6-6/10/2011
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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
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WEEK 2: 6/13-6/17/2011
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I was at SB5.0 from 6/14-6/18 and was mentoring remotely via email.
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WEEK 3: 6/20-6/24/2011
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I was away 6/22-6/25 and mentored remotely via email.
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WEEK 4: 6/27-7/1/2011
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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 to generate and test a new liquid handling class for the robot so we can accurately pipet enzymes in glycerol.
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WEEK 5: 7/4-7/8/2011
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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.
Doug, Swapnil, Suma and I also had a teleconference with Josh Gilmore to discuss using invertases in our iGEM project this summer and to get his expert opinion on the invertase design scheme we came up with.
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WEEK 6: 7/11-7/15/2011
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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.
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WEEK 7: 7/18-7/22/2011
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Transformations with ligations using the new chemically competent cells from Bioline worked extremely well. After testing transformations with both 50uL and 25uL of cells, both worked very well with 100+ colonies on the 25uL plate and more than double on the 50uL plate. Since the 25uL plates worked so well, we cut the cell volume from 25uL for the remainder of our reactions in order to make the competent cells last longer and thus cut down on cost.
I ordered 96-well plates for the robot (Eppendorf Twin Tec Full Skirted PCR Plates) and 6 more boxes of chemically competent cells from Bioline in the hopes that they will last through the end of the iGEM project.
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WEEK 8: 7/25-7/29/2011
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I met with Swapnil and Evan, a graduate student, to discuss our plans for characterizing the simple devices the iGEM team is building using the FACS machine. Due to the delicate nature of the FACS machine, Evan and I will be running the FACS analysis for the iGEM team to avoid having multiple untrained people working on the machine.
I also met with Evan separately to come up with the detailed protocol we will follow for running FACS samples.
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WEEK 9: 8/1-8/5/2011
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The Wellesley team members joined us on Monday to demonstrate their new software tool called Gnome Surfer Pro. The wet team members were split into two groups and participated in the demo and gave feedback.
The wet team also participated in the BU comp team members' Clotho Dojo. The Clotho Dojo ran for the entire day and was used to demo the new applications the computational team had designed in Clotho. We also gave immediate feedback on the apps they developed and offered suggestions for improvement for use in the wet lab.
I also met with Doug, Swapnil, Jenhan and Craig to go over some very useful invertase information that Josh Gilmore had sent us previously. We used our meeting to ask each other questions and to clarify how invertases work and what other applications they may be useful for.
Given the lack of success using SBFP2 (BBa_K156010) and it's poor gel results listed on the registry, we requested a new part from the registry (BBa_K156025) which contains RBS+SBFP2 (B0034+K156010).
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WEEK 10: 8/8-8/12/2011
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Induction of pBad continued to fail despite adding up to 5mM arabinose to the cultures. We discovered that the pBad promoter (BBa_I13453) we were using required AraC to work and that our commercially purchased cells lack the AraC gene. To remedy this error, we have started working with BBa_I0500, a pBad promoter that also contains the AraC gene.
I met with Swapnil, Suma, Evan, Jenhan and Craig to brainstorm new ideas for using invertases/recombinases beyond the scope of what we originally designed at the start of the summer.
Chris Anderson came to visit and we met with him to discuss our iGEM project and other work.
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WEEK 11: 8/15-8/19/2011
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We had a final team meeting with the BU members to wrap up the summer and generate to-do lists for finishing the work.
The major wet lab tasks included submitting finished devices to MIT, working on the outreach plan and finishing the lab notebooks.
We also met with Orit and Consuelo to start planning the presentation for the regional jamboree. We also planned a weekend-long final "bootcamp" for the students to finish the wiki and tutorial/demo videos and to work on the presentation and poster. It will take place at Wellesley the weekend of September 10th.
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WEEK 12: 8/22-8/26/2011
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During the final week of iGEM, I made sure videos were taken in the wet lab and lab notebooks were up to date. I also asked the wet team members to make a list of devices they completed that were going to be submitted to the registry.
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