Team:Austin UTexas/Project/Plasmid Study



Spring Protocol


Plasmid Construction and Transformation


Plasmids were constructed with standard BioBrick cloning procedures and enzymes. Composite plasmids were constructed using the following unique BioBrick combinations:


Plasmid Fluorescent Protein BioBrick Fluorescent Protein Promoter/RBS BioBrick Promoter;RBS Strength
1 BBa_K592100 BFP BBa_K608004 Strong; Weak
2 BBa_K592100 BFP BBa_K608007 Medium; Weak
3 BBa_E0020 ECFP BBa_K608007 Medium; Weak
4 BBa_E0020 ECFP BBa_K608003 Strong; Medium
5 BBa_E0020 ECFP BBa_K608002 Strong; Strong
6 BBa_K592101 YFP BBa_K608002 Strong; Strong
7 BBa_K592101 YFP BBa_K608006 Medium; Medium
8 BBa_K592101 YFP BBa_K608007 Medium; Weak
9 BBa_K864100 SYFP2 BBa_K608002 Strong; Strong
10 BBa_K864100 SYFP2 BBa_K314100 Strong; Very Strong
11 BBa_K864100 SYFP2 BBa_K608006 Medium; Medium
12 BBa_E0030 EYFP BBa_K608007 Medium; Weak
13 BBa_EE030 EYFP BBa_K608006 Medium; Medium
14 BBa_E0030 EYFP BBa_K314100 Strong; Very Strong


Several fluorescent protein/promoter+RBS combinations were studied in more than one experiment, which is why each plasmid above is classified as a 'unique' combination.

The BioBricks in each pair were ligated together using T4 Ligase, and transformed into TOP10 E. coli. Prepared cultures were streaked onto LB/chloramphenicol (CAM) agar plates, and were placed in a 37°C incubator overnight. After at least 20 hours, the plates were retrieved,and the one exhibiting most fluorescent colonies and for each team member participating in the experiment was selected. Three independent, fluorescent colonies were selected and each placed into 5mL of LB/CAM media. The tubes for each were labelled with the date, the culture name, DAY 1, and the strain used. The tubes were placed in a 37°C shaker-incubator and the plates were placed in a 4°C cold room for storage.



Fluorescence Readings

Fig.2 ^^^REPLACE WITH BETTER RESOLUTION



The primary aim of this project was to assess the fluorescent protein coding sequences that are most prone to breaking/mutating. In order to do this, we had to propagate different fluorescent proteins and record their fluorescence daily.

The day immediately following the start of the DAY 1 cultures, the DAY 1 cultures were retrieved and resuspendeded. Three empty culture tubes for each participating team member were collected and labelled the same way as the first set of culture tubes, but with DAY 2 instead of DAY 1. 5mL of LB/CAM media were placed into each culture tube, and 5μL of culture from the microcentrifuge tubes were pipetted into the appropriate culture tube. The culture tubes for DAY 2 were placed into the shaker-incubator, and the DAY 1 culture tubes were placed in the cold room.

200μL of each DAY 1 culture were pipetted into a well on a 96-well plate (which was stored on ice when not in use). The well number, date, and culture name were recorded, and the plates were read for fluorescence the following morning. This process of moving cultures forward and obtaining fluorescence readings was repeated daily until the culture fluorescence dropped dramatically, or until DAY 10, so that each culture had a maximum 10 culture tubes over the 10 days.


The DAY 10 cultures were allowed to incubate and were sent for sequencing. A full "control" plasmid sequence was created for each culture by inserting the recorded BioBrick sequences into an existing pSB1C3-circular sequence on Benchling.com. The sequencing results were uploaded to Benchling and compared to the predicted original sequence of the plasmid for any possible mutations.



summary figure(?)



Summer Protocol


Assessing Yellow Fluorescent Protein genes for stability


Four strains of E. coli were selected to be transformed for the:Top-Ten, MDS-42, BL-21 (DE3), and BW-25113. These Top-Ten, BL-21 (DE3), and BW-25113 were all selected because of the fact that they are commonly used strains in research of synthetic biology. The MDS-42 strain was selected because that strain has many of it’s IS elements removed from its genome. Previous research in the spring has shown that a major cause of mutation in genetic devices is transposable elements inserting into the plasmid. So the hypothesis is that the MDS strain will exhibit greater longevity in terms of fluorescence than the three other strains.

All four strains were transformed with three plasmids that were designed and constructed in spring of 2015. The plasmids contained the same backbone (pSB1C3) and the same vector- a medium promoter and ribosome binding site. The three plasmids differed in only the type of yellow fluorescent protein used: Yellow Fluorescent Protein, Super Yellow Fluorescent Protein, and Enhanced Yellow Fluorescent Protein. These three were selected because of the variance in stability patterns they expressed in the spring semester.
Fig.1 description here
After each strain was transformed with each plasmid, they were grown into over-night culture tubes and then preserved in glycerol stocks. Mbr>


After some time, the four stocks with the Super-Yellow Fluorescent Protein were grown in media in replicates of six. There were twenty four samples in all. Each culture was grown overnight, a sample was used to carry forward to the next day, a sample was taken for flow cytometry, and glycerol stocks were made. Samples were taken to be analyzed for remaining fluorescence using flow cytometry. Re-suspension of each culture using PBS preceded the use of the flow cytometer. Each sample was then pipetted into a well in a 96-well plate, with every six samples separated by a well filled with PBS only. The flow cytometer reads to fluorescent value of each well by sipping each well automatically using a syringe. The media flows from the syringe and into cytometer to be passed through a laser which counts the number of cells (called events) and the intensity of the fluorescence and repeats this for each filled well. The first three days of samples that were read using flow cytometry are seen in Figure 1. The x-axis is the magnitude of fluorescence, which is using a logarithmic scale. The y-axis is the count of cells or objects in sample with a particular fluorescence. By looking at the counts of positive fluorescence from day to day, it is clear that the Top-Ten strain is quickly breaking down, while the MDS strain has remained stable. After four days, the Top-Ten group of SYFP appeared to have a population that was mostly none fluorescent (SEE Figure 2). On the sixth day, all of the MDS strains, the third and fifth BL-21 strains, and the second BW-25113 strain were carried forward and recorded using the flow cytometer.