To design and create a plasmid that, in the presence of TPA, will create a detectable fluorescent signal. Since TPA is a monomer product of the degradation of PET, optimizing the dynamic range of this signal will allow us to accurately measure PET degradation.
Our team tested a variety of parts to design, build, and test four different plasmids. We designed our constructs based on previous data from the Badran lab that showed only a modest increase in fluorescent signal when TPA was titrated into cell culture. Each plasmid and assay are described below.
Initially, our plan was to experiment with MucK, found in reference 1, as a new importer to test if the importer previously used, tpaK, was limiting or non-functional. We also decided to test the effects different fluorescent reporters might have, and compared mScarlet to mEmerald to see which would produce a better signal. To do this, our team conducted an assay, testing combinations of mucK importer plasmid with reporter plasmids encoding either mScarlet or mEmerald.
Above is plasmid pIGEM1. This is the plasmid map of the first plasmid our team made. It has the antibiotic resistance marker bla (AmpR) for carbenicillin to allow selection for the plasmids that have been correctly constructed, the mEmerald fluorescent protein, TpaR-Regulated Promoter (found in reference 2) as the promoter driving expression of TpaR and mEmerald, the origin of replication RepA and pSC101 to allow for plasmid propogation, transcription factor of RHA1 or tpaR (found in reference 2), and rrnB1 terminator. The part our team was interested in for this plasmid was mEmerald.
Above is plasmid pAB132e18 and is very similar to pIGEM1. It has the same antibiotic resistance marker bla (AmpR) for carbenicillin, promoter of TpaR-Regulated Promoter, origin of replication RepA and pSC101, transcription factor RHA1 or tpaR, and terminator of rrnB1. The key difference is instead of mEmerald, the plasmid has mScarlet, which is the part our team is interested in.
Above is plasmid pIGEM2. This plasmid has MucK as its importer of TPA, ColE1 and Rop as its origin of replication, protein lacI and promoter pLacZ to control MucK, and antibiotic resistance marker aadA to confer spectinomycin resistance. The part of interest here is importer MucK. Below are graphs of the results from the first assay. These graphs and their respective data tables can also be found in the lab journal.
Graph 1: Average mScarlet Signal/OD for all 4 clones
Graph 2: Average mEmerald Signal/OD for all 4 clones
From these graphs, our team noted that the largest fold increase was roughly 2.25 for mScarlet with MucK and roughly 1.77 for mEmerald with MucK. While these increases are noticeable, it is not large enough for a robust biosensor. Furthermore, the background signal and how it increases in signal despite the absence of IPTG, a small molecule that binds to pLacZ to allow expression of MucK and thus TPA detection, does create some concern. Therefore, our team looked at other potential parts that could be inhibiting TPA detection. At this time, our team noticed that a promoter called pTPA3, also found in reference 1, worked decently well. Our team compared pTPA3 to TpaR-Regulated Promoter and found three modifications that could heighten our fold-increase: remove a native RBS site found in the TpaR-Regulated Promoter, alter the -35 and -10 sigma factor sections of the TpaR-Regulated Promoter sequence to match that of pTPA3, and entirely replace TpaR-Regulated Promoter with pTPA3. Our team then decided to conduct a second assay to compare these three modifications. Since the importer and reporter had to be kept constant in this assay and mEmerald with MucK and mScarlet with MucK performed similarly, our team just chose to use mEmerald with MucK. Since mEmerald came from pIGEM1, the first modification (removing the native RBS site) was called iGEM1a, the second modification (altering -35 and -10 sigma factors) was called iGEM1b, and the third modification (replace with pTPA3) was called iGEM1c. All the sequences and sequence changes can be seen below. In the parts page, iGEM1a was renamed TpaR-Regulated Promoter1.1, iGEM1b was renamed TpaR-Regulated Promoter1.2, and iGEM1c is pTPA3.
Below is the sequence of TpaR-Regulated Promoter (labeled by the purple bar), which is divergent and bifunctional.
Below is the sequence for modification iGEM1a. Notice the missing “aggaga” (top) and “tcctct” (bottom) from the original TpaR-Regulated Promoter at the end of the sequence.
Below is the sequence for modification iGEM1b. Notice how the original -35 sigma factor of “ttgaca” (top) and “aactgt” (bottom) and -10 sigma factor of “cttaat” (top) and “gaatta” (bottom) were changed into the pTPA3 -35 sigma factor of “GTACAC” (top) and “CATGTG” (bottom) and -10 sigma factor of “TACAAT” (top) and “ATGTTA” (bottom).
Below is the sequence for modification iGEM1c. Notice how the entire TpaR-Regulated Promoter is gone and pTPA3 takes it place instead.
Below are graphs from the second assay. These graphs and their respective data tables can also be found in the lab journal.
Graph 3: Average Fluorescence/OD for Each Modification at Various TPA Concentrations with IPTG
Graph 4: Average Fluorescence/OD for Each Modification at Various TPA Concentrations without IPTG
In Graph 3 and Graph 4 all 3 modifications are being compared to each other with and without IPTG. In the graph without IPTG, the signal remains relatively constant for all modifications, but modification iGEM1b did exhibit some increase in signal. In the graph with IPTG, the signal for modification iGEM1a essentially did not change and modification iGEM1c decreased, but modification iGEM1b clearly exhibited a noticeable increase in signal which is what our team was hoping for.
Graph 5: Comparing Signal of iGEM1b with and without IPTG at Various TPA Concentrations
This graph focuses on only modification iGEM1b with and without IPTG. A clear dose response to increasing TPA concentrations and an IPTG-dependent boost in the signal can both be observed, suggesting that MucK is functional. However, the fold-increase comes out to roughly 1.82, which is noticeable but not as high as our team would have hoped for it to be.
Graph 6: Comparison between mEmerald From First Assay and iGEM1b From Second Assay
TThis graph compares the second assay to the first assay. Both “mEmerald Assay” and “iGEM1b assay” share the same plasmid parts but differ in the promoter, with the former having the TpaR-Regulated Promoter and the latter having the altered -35 and -10 sigma factor. From this graph, it can be seen that the modification did improve the fold-increase, as the first assay had a fold-increase of roughly 1.77 and the second has one of roughly 1.82; however, these values for a biosensor are still not the most ideal. Beyond fold increase, the modification also noticeably widened the gap between the plasmids with and without IPTG, which was definitely something our team was hoping to see.
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