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Team:BostonU/Data
From 2013.igem.org
Data Collected
New Part Creation
We made new MoClo parts by modifying existing Biobrick plasmids from the Distribution Kits. We designed PCR primers to add Type IIS Enzyme sites and fusion sites to flank the 5’ and 3’ sides of the coding sequence. The PCR products were gel extracted and inserted into a Level 0 backbone with matching fusion sites in a MoClo digestion-ligation reaction.
The reaction products were transformed into competent cells and plated with IPTG and X-gal for blue-white screening.
White cultures were grown up in LB Broth with chloramphenicol. The following day the cultures were spun down and the plasmid extracted via the miniprep protocol. The level 0 parts were sent for sequence verification and cultures were archived as glycerol stocks in -20 degree Celsius and -80 degree Celsius freezers.
From the collection of Level 0 parts, we designed level 1 constructs and combined level 0 parts in a MoClo digestion-ligation. The reaction product was transformed and plated with IPTG and X-gal for blue-white screening. Selected colonies were grown up in cultures and the plasmids extracted the following day via miniprep protocol. Due to the size of the level 1 parts that were made (~900-1200 bp), the plasmids were digested and analyzed with gel electrophoresis. Level 1 Parts were also sequence verified if the gel results matched expected sizes.
The process was repeated with Level 1 parts as reactants to make Level 2 products, such as the inverter.
To see the complete list of parts we made this summer, please refer to our Parts Submitted page.
Characterization of Level 1 MoClo Devices
Using BBN Technologies TASBE tools under the guidance of Dr. Jacob Beal, we analyzed our library of constitutively expressed RFP Level 1 MoClo devices.
Below, you can see more details of our experimental design and the controls we used, which are required by the TASBE tools in order to convert the arbitrary fluorescence units obtained from the flow cytometer into absolute units in the form of molecules of equivalent fluorescein (MEFL). This allows the user to show their data in absolute units that then allow scientists to compare experiments across labs and machines.
The Cytometer Setup and Tracking beads offered by BD Biosciences were utilized to set the laser delay and optimize the cytometer settings prior to running any samples through the Fortessa.
We also used Spherotech's 8-peak particles (RCP-30-5A) in order to obtain standard MEFL units for the FITC channel. They are also used to measure the long term performance of the flow cytometer and should be included in every experiment run through the flow cytometer.
In order to obtain MEFL measurements for the RFP protein, we had to utilize a dual positive control that had a FITC channel fluorescent protein. We used YFP for our FITC control. Below is a histogram showing the co-expression of RFP and YFP in our device, as shown. We used the J23014 promoter in both devices, along with the BCD2 5'UTR element. The histograms show the similarity of expression between the two colors on our machine and the dot plot shows a agreement between them. The bottom figure is from the TASBE tools Color Translation model, which agrees with our dot plots and histograms.
Below is a subset of our results that demonstrate the strength of the TASBE tools for analyzing fluorescence data. (We will be showing the full set of data at the Jamboree.)
Here we are showing RFP fluorescence as both arbitrary units (top graph) and in MEFLs (middle and bottom graphs). All of the devices shown were made using the MoClo method. However, we converted a handful of our Level 1 devices into a BioBricks compatible format for the iGEM competition requirements. Our original MoClo parts (BBa_K1114500, BBa_K1114502-4) are in the pSB1K3 backbone while the BioBrick switches (BBa_K1114701-4) are in the pSB1C3 backbone, as required for the competition submissions.
At first glance, the top graph showing arbitrary units suggests that the expression levels between the two backbones varies. However, when we convert our data into MEFLs using the TASBE tools, the data is binned into subpopulations based on fluorescence expression amounts. This binning allows us to see that these subpopulations within each sample that have different levels of expression. If we only look at the geometric mean of the entire population, then we are missing parts of the data that can help explain unexpected results.
We will continue to characterize more parts prior to the Jamboree and look forward to showing more complex devices in our presentation.
Datasheet Tool
The Datasheet Tool was coded primarily in Java for the back-end server side and in HTML from the front-end client side. The app was coded in the NetBeans IDE and we stored all of our code on a private GitHub repository.
Datasheet User Interface
This section allows the user to fill out specific design information.
Eugene Results
We wrote Eugene scripts for our projects this summer. The wiki upload option does not allow .eug Eugene files so we uploaded our code as plain text files. To run our code in the Eugene Scriptor, simply save the code as .eug.
The pConstitutive Library Eugene file shows all of the constitutive promoter and fluorescent protein permutations using the Anderson promoter library.
The pRepressible Library Eugene file shows all of the possible repressible promoter and gene relationships based on our MoClo library.
The Quorum Sensing Eugene file shows the permutations possible with the specified rules, such as the repressing relationship between certain promoters and genes and the small molecule arabinose inducing the pBad promoter.
