Designed by: Astrid T�bber Group: iGEM19_Bielefeld-CeBiTec (2019-10-15)
f1_ori_mCherry-M13K07-pVIII-fusion_pIII
This plasmid includes an f1 origin of replication, the fluorescence marker mCherry, the genes VIII and III from the M13K07 helperphage and the iGEM backbone pSB1C3 with a chloramphenicol resistance.
Sequence and Features
Assembly Compatibility:
10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 126
1000
COMPATIBLE WITH RFC[1000]
Assembly of Plasmids
We obtained the parts of gene II-VIII, gene III-IV, truncated gene III, terminator, and mCherry-VIII gene through PCR with customized primers.
After proving the accuracy of the PCR product, we assembled the parts together through gibson assembly and transformed the assembled parts in E. coli DH5 alpha.
After conducting growth experiments, we isolated the proteins and took them for a series of analysis to prove the existence of the parts in the assembled system.
Fig. 1. Result of supernatant PCR performed with primers which specifically amplify a region on the application plasmid.
The PCR yielded a product only on the supernatant of the culture containing E. coli transformed with the Assembly Plasmid and the Application Plasmid. In the culture of E. coli only with the Application Plasmid no PCR product is visible. This demonstrates, that E. coli with both plasmids produces our Troygenics and sets them free in the supernatant. So, their DNA is also detectable in the media. The missing PCR product in the sample from the culture with only the Troygenics DNA shows that this plasmid alone is, as expected, not sufficient to produce the Troygenics. Furthermore, this results shows, that occurrence of the PCR target inside E. coli cells does not interfere in this assay and produce false positive results. Therefore, this assay is an easy detection of Tryogenics production, assembly and export by our two-plasmid system.
Fig. 2. Absolute quantification of application plasmid, assembly plasmid, and genomic E. coli DNA by ddPCR. Troygenics were purified, the DNA was isolated and diluted and ddPCRs specific for the application plasmid, the assembly plasmid and the E. coli genome were carried out. The individual droplets with PCR product (blue) and without PCR product (black) are shown for the three primer pairs.
After we redid the Troygenic purification with the expanded protocol from NEB, we did the ddPCR again.
This time there was even less E. coli DNA in the sample than before. The application plasmid is 4.1- and 21.4-times more common than the assembly plasmid or genomic E. coli DNA respectively.
This experiment demonstrates, that the new protocol resulted in Troygenics with a higher purity. Additionally, the new protocol yielded a higher amount of purified Troygenic DNA.
Fig. 3. Relative amounts of application plasmid, assembly plasmid and genomic DNA from E. coli in the Troygenic DNA purified with the initial protocol
Fig. 4. Relative amounts of application plasmid, assembly plasmid and genomic DNA from E. coli in the Troygenic DNA purified with the extended protocol from NEB
Fig. 5. Fluorescence of a cultures of E. coli ER2566 transformed with both of our plasmids and mCherry as a comparison measured with TECAN infinite M200 plate reader
Our basic-version of the Troygenics presents mCherry on their coat. This means that we can detect correct Troygenics assembly by measuring the fluorescence of the incorporated mCherry. We measured the fluorescence on the TECAN Infinite M200 plate reader, for which we used Texas Red 2.5 µM as reference dye for our Troygenics. In a first experiment the excitation wavelength was 570 nm and the emission wavelength 610 nm. We were able to detect fluorescence caused by our Troygenics.
In a second experiment we recorded an emission spectrum of our Troygenics (Fig. 5). As a result, we have seen that there was a typical emission graph with a peak around 630 nm. The emission intensities of wavelengths between 550 and 600 nm are not shown because they relate to the excitation wavelength.
We compared the emission spectrum of our Troygenics with the emission spectrum of mCherry, because the fluorescence marker cloned on the Troygenics is mCherry, too. We have seen a difference in the emission peak of about 20 nm. This is probably because the mCherry on our Troygenics is fused to the whole Troygenic. Because of this, there might be a different folding resulting in a shift of the emission spectrum.
