Team:Austin UTexas/Project/Plasmid Study
Measuring Fluorescent Protein Gene Stability
Introduction
One of the major issue facing genetic engineering is the overall longevity of genetic devices once inserted into organisms. An organism can be modified, but that does not ensure that the modification will last. Subsequent generations of modified organisms often lose or “break” the genetic device through mutations. Mutations that cause the inactivation of a plasmid or an inserted device decrease the metabolic load of that organism and allow the mutant and its descendants to replicate and grow faster than the originally modified organism, allowing the “broken” mutant to overtake a population.
The stability of the sequence is the focus of our research over the course of the summer. We wish to identify sequences that contribute to the relative instability of genetic devices so they can be used to predict the longevity of a genetic device as well as be taken into account when creating and modifying such devices. In the spring of 2015, E. coli was transformed using various fluorescent genes and monitored for breakage, with varying results depending on the plasmid used. Three plasmids were selected from the spring experiment to be used. Each plasmid differed by the fluorescent protein used: Yellow fluorescent protein (BBa_K592101), Super-folder Yellow fluorescent protein (BBa_K864100), and Engineered Yellow fluorescence protein (BBa_E0030).
Over the summer, these three plasmids were used to transform four strains of bacteria (TOP10, MDS42, BL21 (DE3), BW25113). Growing these transformed strains in culture and monitoring their fluorescence and stability will hopefully elucidate more regarding patterns in the sequences that contribute to the instability of genetic devices. Understanding this could lead to more stable devices and thus could increase the longevity of genetically modified organisms.
Since genetic stability can be problematic for any work involving synthetic biology, we wanted to start by determining what sequence motifs make a genetic device unstable. So, in the spring, we began an experiment to study genetic instability of different plasmids in E. coli. We designed and transformed 14 plasmids into TOP10 E. coli. Then, we propagated the strains until either fluorescence was lost or the experiment was concluded.
Methods
Plasmid Construction
Each plasmid comprised of a [http://parts.igem.org/Part:pSB1C3 pSB1C3] backbone, which included a gene for chloramphenicol resistance and has a copy number of 100-300. Further, each plasmid received a promoter, ribosome-binding site, and coding sequence for a fluorescent protein. Plasmids were constructed with standard BioBrick cloning procedures and enzymes. Composite BioBricks were constructed using the following unique BioBrick combinations.
Fig.1:
| 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 DNA 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 a single colony was grown overnight in 5 mL of LB/CAM media. After sequence confirming the plasmid in each culture, we froze a fraction of these cells in glycerol at -80° C for storage.
Fluorescence Readings
After we transformed the plasmids into E. coli, we were almost ready to begin the propagation phase of our stability experiment.
First, we steaked transformed cells for each plasmid onto LB/CAM plates and let them incubate overnight. The next day, we selected a number of independent colonies that were still fluorescing for each plasmid. Then, we placed the colony in 5 mL of LB/CAM media and grew each overnight in the shaker at 37° C and 215 RPM. We refer to these cultures as 'Day 1'. The next day, we began the propagation phase of our experiment.
Moving forward, each day we retrieved the overnight cultures from the shaker. We used 5 μl to start a fresh 5 mL overnight culture with LB/CAM media, which was then grown overnight. Next, we measured fluorescence using a dark reader by pipetting 200μL from each culture and into a well on a 96-well plate. Finally, the day's cultures were placed in the cold room for storage. This process was repeated until either fluoresence was lost or ten days had past.
After the propagation phase, we sequenced the final cultures and compared this sequence to the original to determine which mutations had dominated the cultures.
Results and Discussion
Each plasmid created had a great amount of variance in their stability and longevity over the course of the experiment depending on the fluorescent protein that was used in the plasmid.
Plasmids that bore the SYFP2 gene tended to be fairly unstable. Strains with SYFP gene tended to break down after around three days, going from very high fluorescence to no observed fluorescence. Furthermore, some samples of SYFP2 lost functionality after merely a day, showing the SYFP gene is very unstable across generations. The SYFP2 plasmids most often accumulated IS inserts, which completely removed the fluorescent protein gene from the plasmid and ceased production the protein. The most common IS elements observed were IS10R and IS10L. Furthermore, S10L/R expressed preference for a particular nucleotide location (GGCGTAGTACC). Two point mutations were also independently observed in two samples of SYFP2.
Plasmids that contain the YFP gene also tended to display instability. Samples with the YFP gene often broke around five days, and a myriad of mutations appeared to have contributed to this instability. IS elements were another common feature among the YFP group. Point mutations in various locations also appeared in the YFP group, such as in the promoter and ribosome binding site. Two samples also featured a large deletion from the plasmid.
The BFP group of plasmids also had moderate instability, similar to the YFP group of plasmids. The longevity of this group of plasmids was around five days before the plasmid broke down. IS elements were very common in the BFP plasmid group along with promoter deletions.
Interestingly, the EYFP and ECFP plasmid groups were incredibly stable, remaining fluorescent for over ten days. Only one sample in the entire EYFP group displayed a mutation, which was a promoter deletion. Only two mutations were shown in the entire ECFP group, a large deletion and an In-del mutation. Overall, these two fluorescent proteins have shown to be very stable (see Table 1 for full results of mutations).
Based on the patterns of mutations, it is clear that IS elements are a major source of instability in plasmids. Furthermore, because of the common location of insertion for IS elements, they must have a sequence preference. By changing the sequence, it is possible to increase the stability of plasmids by eliminating a mode of mutation. Furthermore, by elucidating the contribution that IS elements make towards the instability, we can better predict and reduce the instability of future plasmids.