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

This year our team has been focusing on creating a system that will allow other iGEM teams to easily control the gene expression speed of an arbitrary protein without requiring a costly wholesale redesign of existing architecture. To that end we characterized how protein degradation tags (pdt) can be used to modularly change the speed of mScarlet-I, a fast folding (7 minutes) high quantum yield red fluorescent protein. We also created a set of easy cloning parts which enable future iGEM teams to clone our parts without needing intermediates. At the UVA Mid-Atlantic meetup, we were particularly intrigued by University of Maryland iGEM’s copper sensor. After discussing with them, we agreed that we would collaborate on improving the function of their copper sensor (BBa_ BBa_K2477013). Since they primarily wanted to use their copper sensor as an educational tool, reducing the time required to get results would help them keep students engaged, as well as enable them to use their device in labs or outreach activities that did not have a lot of time.

To help them increase their gene expression speed we proposed that we jointly create and characterize modified versions of their parts with pdts. Since time was short and Maryland did not have access to a cell sorter, we simply had them ship us their parts, and then did the modifications and characterizations at William and Mary, before sending the modified parts back for use. On the other end, William and Mary sent blinded speed constructs to Maryland for testing. We hoped that this would enable us to confirm degradation in platforms other than our own, and potentially show speed change as well.

We transformed the plasmids (listed below) resuspended from the Distribution Kit into E. coli DH5-alpha cells. Colonies were given 16 hours to grow.

Calibration

Before we started plate reader measurement, we obtained the OD600 reference point and the fluorescein fluorescence curve in the microplate reader to standardize the absorbance reading and cell-based fluorescence reading. Our model was a Synergy H1 Hybrid Multi-Mode Microplate Reader. Ludox-S40 silica nanoparticles were used to calculate the correction factor of OD600. Black 96-well plates with clear bottoms were used. For the plate reader our excitation and emission setting were 485 nm and 528 nm respectively (Same setting was used for all experiments below).

The dilution curve of fluorescein was performed by carrying out a 11-step, 2-fold serial dilution of green fluorescein. Final scaling level was determined from medium-high points in the dilution that is likely to be less impacted by saturation or pipetting error. The μM Fluorescein/a.u.is defined as mean of mid-high level fluorescein concentration divided by the obtained plate reader reading.

Cell Measurement

2 colonies of each device was inoculated over night into 5 ml Luria- Bertani medium with 25 μg/mL Chloramphenicol in a 37°C, 220 rpm shaking incubator. Cell cultures were diluted to a target OD600 of 0.02 into same LB medium in 50 mL falcon tube covered with foil before use Diluted cultures were further grown at 37°C and 220 rpm. At 0, 2, 4, and 6 hours of incubation, 500 μL aliquot was taken from each two colonies of the 8 devices and were placed immediately on ice to prevent further growth. At the end of sampling point, 4 replicates 100 μl of each sample was pipetted into a 96-well microplate with the arrangement as below. Data was imported into the Excel Sheet for submission.

Below is the Fluorescein Standard Curve we obtained, from which we can still see the problem of saturation. We also converted the calibrated data of the time-measurement into a uM Fluorescence a.u./ OD600 versus time graph. Besides Device 1 and Device 4, all the others constructs show consistency of standardized fluorescence level in the two colonies over time.

From our experiment, we conclude that that BBa_J364100 is a stronger RBS, with an increase of 32.0%, 74.2% and 16.2% expression under J23101, J23106 and J23117 respectively compared to BBa_B0034.

The Standardized RBS tested in this experiment, BCD (bicistronic design) 2 is a synthetic cistron leader peptide region that contains two Shine Dalgano sequences that is reported to have increased precise and reliable translation initiation [3]. Device1 and 4, 2 and 5 and Device 3 and 6 features the same strong (J23101), medium (J23106) and weak (J23117) promoters from the well-characterized Anderson promotor family in iGEM registry. Device 1-3 are under standard RBS BBa_B0034, (which William_and_Mary iGEM 2016 has proudly characterized), while Device 4-6 incorporate the test subject BBa_J364100 (BCD2).

Since all of the devices are under constitutive promoters, we assumed that fluorescence expression to be consistent over time in an optimal growth condition (37°C in LB medium). we compiled a total of 48 data of all 4 time points and 2 colonies of the same RBS and did an anova test for BBa_B0034 and BBa_J364100 and obtained a p-value of .085.

The failure of getting a significant difference between groups may be due to a small sample size and limitation of place reader measurement. Since Device 1 and Device 4 accounts for most of the variation, and both of which are under the same promoter, another possible explanation would be the context dependent performance of J23101, and an insulator part may be needed to further investigate property of this RBS if the same problem occurs across different teams [4]. We thank the iGEM Measurement Committee again for providing us an excellent opportunity to be part of this study and look forward to see the study results when data from all participating teams are put together.

[1] Kwok, R. (2010). Five hard truths for synthetic biology. Nature, 463(7279), 288-290. doi:10.1038/463288a [2] Beal, J., Haddock-Angelli, T., Gershater, M., Mora, K. D., Lizarazo, M., Hollenhorst, J., & Rettberg, R. (2016). Reproducibility of Fluorescent Expression from Engineered Biological Constructs in E. coli. Plos One, 11(3). doi:10.1371/journal.pone.0150182 [3] Mutalik, V. K., Guimaraes, J. C., Cambray, G., Lam, C., Christoffersen, M. J., Mai, Q., . . . Endy, D. (2013). Precise and reliable gene expression via standard transcription and translation initiation elements. Nature Methods, 10(4), 354-360. doi:10.1038/nmeth.2404 [4] Davis, J. H., Rubin, A. J., & Sauer, R. T. (2011). Design, construction and characterization of a set of insulated bacterial promoters. Nucleic Acids Research, 39(3), 1131–1141. http://doi.org/10.1093/nar/gkq810