Difference between revisions of "Team:Austin UTexas/Project/Plasmid Study"

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.

Fig.1 description here

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.

Spring 2015 Data and Observations

Data from the first experiment gave a strong indication as to whether or not a coding sequence was stable. Specifically, plasmids containing coding sequences for BFP, SYFP2 and YFP broke swiftly while coding sequences for EYFP and ECFP remained mostly intact by the experiment’s conclusion. Several types of mutations were observed: IS element insertions*, point mutations, various deletions, and phage insertions. IS element insertions were the most common mutation, and occurred exclusively in the ‘unstable’ plasmids (BFP, SYFP2 and YFP). 23 of 40 independent cultures for these coding sequences exhibited a loss of fluorescence due to mobile element insertions. The next most common mutation were single nucleotide polymorphisms; 6 point mutations were observed. Four of these point mutations occurred in a promoter (which?), while one was located in an RBS (which?) and one altered a start codon. Further, five samples incurred promoter deletions (which promoters?), and five additional samples developed miscellaneous large and small deletions. Finally, three samples with coding sequences for BFP contained the same phage element, suggesting that this event occurred before independent colonies were selected. (possibly add some discussion about promoters—repeat mediated?) Our results displayed an array of mutations, and potential experiments for follow up. We decided to focus on IS element insertions, which were most prevalent. These insertion elements are found in the genome of Top10 E. coli. They replicate ~independently*? and insert themselves into different DNA locations. If this DNA location happens to be within the inserted plasmid, the genetic device breaks, granting a depreciated metabolic load in the mutant and reducing fluorescence. A number of mobile elements in E. coli have been identified. In particular, we observed mobile elements IS10L, IS10R and IS1, with the latter occurring only once*. Interestingly, in plasmids with YFP and BFP, these IS elements exhibited only moderate insertion preference. In these sequences, IS elements were found throughout both promoters and coding sequences. However, in SYFP2 plasmids, IS10L/R expressed preference for a particular nucleotide location (GGCGTAGTACC) near the start of the SYFP2 plasmid. (same nucleotide sequence in each plasmid?)* Based on this preliminary data, we consider coding sequences for BFP, SYFP2 and YFP to be unstable while we consider coding sequences for EYFP and ECFP to be stable. Consequently, we decided to focus on SYFP2 as we continued studying instability.

(WILL BE MOVED TO FIRST DATA SET) Observations and Discussion

insert mutations in SFYFP
EYFP, CYFP stable
what contributes to stability
what does this mean about what sequences are stable</br>
next steps