Difference between revisions of "Team:William and Mary/Notebook"

Starting off the summer by cloning parts that will enable us to test the basic functionality of the mf-Lon for protein degradation. These parts consist of pBad and J231** parts for all of the sfGFP pdt tag variants, as well as mf-Lon. Currently we have confirmed versions of 3 of 6 J231** sfGFP variants, as well as an mf-Lon under the control of J231**.

We began to lay down the foundation for our big outreach project, which will become a searchable database of past outreach projects from award-winning teams. Project categories are being brainstormed in addition to tags for different audiences.

Continued to make progress towards functionally validating and testing our mf-Lon and pdt parts. While our pBad sfGFP pdt tags have been lagging behind, we moved ahead with making combination parts for our simple circuit (LacI pdt and pLac sfGFP pdt combos), and we have been validating our existing mf-Lon and pdt tagged sfGFPs. From the current data, it appears that some of our sequence confirmed pdts are working, while others are not. However, all of our mf-Lons are functional.

We entered information about 2016 and 2015 IHP/EPE award-winners and decided to move the database information to a spread sheet to make it easier to eventually create a website. Alyssa met with Professor Griffin, who specializes in community-based research and outreach. We worked on the PowerPoint and brochure for our Bioengineering Lab, and the PowerPoint is now playing and visible to anyone passing by our lab. GSCC got back in touch with Alyssa on possibly organizing a field trip for Girl Scouts to the BEL. In addition to that, Alyssa communicated with Kelley Clark from the STEM Education Alliance to organize education activities with AP Bio/9th Grade Bio students in the fall.

This week, we were held up a great deal due to not being able to send for sequencing. We are currently in experiments to confirm pdts are working, and cloning inducible sfGFP pdt parts to be able to measure speed. We are switching over to pLac instead of pBad, which entails additional concerns due to the need for a repressor.

Alyssa got in touch with Jacqueline from GSCC to discuss doing activities with the Girl Scouts. We will host about 50 girls on two Saturdays either in September or October. For the database, we've gone back to 2011 outreach projects but will likely end up going back to 2008. Alyssa started organizing a speaker series, and three professors have agreed to speak. Theresa and Alyssa started working on a video for our project. Sejal finished updating the K-5 and 6-8 activity booklets.

We finally confirmed an mf-Lon and pdt combo. We currently have characterization of J23100 tags along with a pBad mf-Lon. We are also trying to functionally confirm a J23116 mf-Lon, and characterize R0011 (pLac0-1) pdts with pBad mf-Lon. One issue we have been running into is with mf-lon causing toxicity/growth inhibition. We will aim to tune our parts around this. The graph below shows evidence for mf-Lon induced toxicity. The J23100 construct does not possess a pdt and fluorescence decreases despite that no GFP is getting degraded.

We set up the dates for the GSCC visit in October. We have continued to enter entries into the database spreadsheet and have decided to house the database in the wiki so it will be accessible to other iGEM teams and iGEM leadership. We made and edited the script for the viedo describing our project. Alyssa recorded the voiceover and Theresa filmed the footage.

This week we focused mainly on two things. Number one is continuing to validate and clone parts. Number two is determining exactly how we plan to measure everything. The first one has made steady progress, (cloning of combination parts, quorum circuits, and functional validation of our pLac pBad circuit), while the second one still requires a great deal more thinking. Namely we have to investigate some claims from a paper, as well as figure out more robustly what we want to measure.

We published the video explaining our project to YouTube and Facebook. We now have two dates locked down for our bioengineering speaker series. Professor Schniepp is speaking on the 13th and Professor Kerscher is speaking on the 20th (4:00-5:00). Sejal and Alyssa continued working on the high school synthetic biology activities booklet.

This week we were focused on trying to measure a functional induction curve with a "steady state" as well as confirming our pdts at the single cell level. We managed to accomplish the latter, though the effect size is not perfect, and there is still a lot of tuning to do. We've also been working on getting ready to use the scope to attain really robust time course measurements.

Made a lot of progress in planning and solidifying plans for education outreach. Alyssa has been in contact with Deanna Marroletti from Camp Launch and she will be visiting with her students on Wednesday, July 19th. We are planning on doing a DNA extraction activity, RFP streak, and gel electrophoresis. We finished the database up to 2008 prize-winning teams. We are now going to go to 2016 nominated teams.

Induction time course for WM17_377 or K2333420 (pTet sfGFP + J23105 TetR) using 30 minute time points. t1/2 to steady state is roughly 30 minutes, which is close to the expected doubling time of cells in our media.

Alyssa had a productive meeting with Kristin Cosby, a teacher from Jamestown High School. We will be working with a group of science teachers on August 31st. We had the first event for our Bioengineering Speaker Series. Professor Schniepp gave his talk and people seemed to enjoy the interdisciplinary nature of it. On Friday, we spoke with students from Plus S and answered questions about majors, iGEM, and research.

Callan did Gibsons for WM17_386 (pLac0-1 mfLon + J23107 LacI). Christine miniprepped sfGFP pdt + pBad mf-Lon parts. Looking at analysis from the flow cytometry (FACS) run for J23100 sfGFP pdt parts, we are indeed seeing growth effect problems, and that's having an impact on fluorescence. We have working pTet pdts. We're geneblocking pLac mf-Lon.

We held the second event for our Bioengineering Speaker Series. About 30 people attended. We also have a potential collaboration going with Senator Monty Mason. He wants to help us with outreach and invited us to a meeting. On Wednesday, we had a great time with two groups of rising 8th graders (16 students in each group). We did 2.5 hour lessons that incorporated strawberry DNA extraction, gel electrophoresis, and RFP streak. On Saturday, we did a presentation for Focusing on the Future, which was a career fair of sorts for the campers.

This week, we ran induction curves on J23100 pBad parts with pdts to try to get confirmation of functionality as well as to determine optimal concentrations. We also ran induction curves on working pTet combo parts but found fluorescence values to differ largely among constructs (could be attributed to bad luck with inoculating things.) We tried to run another time course to see if the time to steady state changes when a pdt + mf-Lon is added, but having weird issues with the FACS clogging--hopefully this can be solved by next week.

We held the third event for our Bioengineering Speaker Series. Dr. Puzey gave a fascinating presentation on how plants can have applications to bioengineering. Dr. Saha prepared delicious "plant-based, mind-altering" food for the audience.

Cloned pLac mf-Lon combo on 1C3 and 3K3. Performed functional tests on pLac parts and re-characterized pdt#3, 3a, 3b, 3d.

Continued adding and editing entries for the database. Alyssa worked on the Bioengineering Lab website.

FACS fixed. Tested different induction conditions for pTet pdt #3, #3a, #3b parts (different concentrations of aTC and arabinose.) We started thinking about how exactly we'd want to design an mScarlet construct. Planning to clone mScarlet versions of J23100 + pTet parts (3K3 + 1C3).

Nova Labs wants a short bio/description of who we are and what we'll be talking about. Looks pretty good.

CiCi got scope to start working over the last couple of days. We got sequencing back and found an error trend in the RBS region of TetR on pTet sfGFP + J23105 TetR (K2333420) variants.

Alyssa and Callan met to plan our Community Lab presentation. Database is rapidly growing.

Cloning J23100 mScarlet pdt parts (K2333414-K2333419). Cici miniprepped and sent out for sequencing.

Alyssa and Callan gave NOVA labs presentation.

Everyone's back in the lab. We did Gibsons for a J23100 + sfGFP pdt#3 (K2333407) re-creation and WM17_301-306 (UNS + pdt). We transformed these in addition to mScarlet-I parts (K2333427-K2333433) for measurements. We ran time courses for mScarlet parts under different concentrations of IPTG, and WM17_377 (K2333420) variants under different concentrations of aTC and IPTG. Data looked messy in general so we're re-cloning mScarlet parts.

Remaking inducible mScarlet control part, WM17_408 (K2333427). Ran an experiment for WM17_401, WM17_394, WM17_396, WM17_397, WM17_398, WM17_399 (K2333413-K2333419) in the plate reader with inducer. Looking at the data analysis, the fluorescence of these constructs is really different from construct to construct (but is consistent between biological replicates.) However, it is very clear that .01mM IPTG has a fluorescence reducing effect, and .1mM even more so.

Ran experiment for aTC-inducible mScarlet pdt parts WM17_409-414 (K2333428-K2333433) in the plate reader. Analysis of data reveals several things. One, all the tags are working. Two, all concentrations of mf-Lon are high enough that we can't see any difference between the different tags. Three, all constructs look to be the same fluorescence. We also decided to FACS mScarlet parts at 4 hours instead of 6: cell density was nice and degradation seems to be occurring. We grew WM17_409 (K2333428) for 5 hours and ran a time course with and without IPTG. Degradation continues after steady state is reached, but it's fairly minor. Callan headed up Gibson cloning of pTet mScarlet + pLac mf-Lon combo parts, and the re-cloning of 408 (K2333427).

This week we ran a 4 hour time course for WM17_409 (K2333428) under aTC and either .1mM IPTG or no IPTG. Every twenty minutes, we took 100µl from each of the tubes and immediately filtered into tubes on ice. We did a second experiment for WM17_410 (K2333429) with the same conditions, and a third experiment for all parts in the series WM17_409-414 (K2333428-K2333433). Analysis showed that pdt#3c part is not working, and our pdts have somewhat different basal fluorescences. Aside from experiments, we did cloning for WM17_415-421.

We did another experiment for WM17_409 (K2333428) under more IPTG conditions. Unfortunately, we didn't see a speed change. Going to try using pdt#3c, since it seems to be weaker than the rest. Ethan designed primers for team UMaryland 2017 and sent out our plasmids to them.

We ran several more experiments this week. The first were for WM17_412 (K2333431) and WM17_410 (K2333429) with either no IPTG or .1mM IPTG. We also did a time course for WM17_411-414 (K2333430-K2333433) under similar conditions. The pdt#3d part worked, and #3a looked quite similar to #3. For the collaboration, we transformed UMD copper plasmids into 10-Beta cells (named the constructs WM17_MD01-WM17_MD06 or K2333437-K2333442) and ran an induction experiment with IPTG and different concentrations of CuSO4.

We hosted our big outreach event on Saturday. We gave a basic synthetic biology presentation in the auditorium and then broke the Girl Scouts off into different age groups to run activities.

Did more inductions for UMaryland parts (K2333437-K2333442). Also ran a 5 hour time course for WM17_409 (K2333428) with and without IPTG. Data for this looked IFFL-like, but we got some incorrect samples at 120 minutes. We decided to do a readjustment attempt with WM17_408 (K2333427). We ended up running experiments for WM17_410, 11, 13, and 14 as well (K2333429-K2333433).

Began to wrap up wetlab with some final experiments. We have seen speed change in pre-induced cells, but we needed to go a little longer in the time course to see steady state. We also organized simultaneous induction for WM17_408-413 (K2333427-K2333432) as well as UMD parts to get IFFL data.

We ran our last two experiments for this year: WM17_408-12 (K2333427-K2333431) and UMD01, 03, and 04. We discovered that pdt #3c does indeed work!