Team:Bielefeld-CeBiTec/Demonstrate

Progress Indicator Animation
Demonstrate
Summary
Our Troygenics are a newly developed, customisable and easy to use system for the specific delivery of any desired DNA to a targeted cell. The results achieved by us show that every part of the Troygenic is working as designed. Firstly the analysis of a template Tryogenic with mCherry represented on its surface showed the correct assembly of the Troygenic. Furthermore, we were able to show specific recoptor-induced endocytosis for S. cerevisiae and A. niger as model organisms. We used S. cerevisiae as a model organism to demonstrate that our Cas13a based CeDIS is working. Additionally, we constructed a microfluidic chip which enabled us to observe our target cells on a single cell level. Finally we assembled a Troygenic which represents ligands for its endocytotic uptake in S. cerevisiae on its coat, and contains an Application plasmid which encodes sfGFP that is regulated by a promotor and terminator for S. cerevisiae. With this Troygenic we showed the successful uptake of our Troygenics by S. cerevisiae.

Troygenic Assembly

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Construction of the Assembly plasmid

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Construction of the template Application plasmid

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Troygenic production and purification

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Demonstration of the correct Troygenic assembly
We have designed and constructed both plasmids needed for the assembly of our template Troygenic. After expression analysis of two different E. coli strains which we co-transformed with the two plasmids, we identified E. coli ER2566 cultivated in baffled flasks as the most promising production strain for the Troygenics.
Firstly we executed a PCR with specific primers for the Application plasmid on the culture supernatant of a Troygenic producing E. coli strain and a strain containing only the Application plasmid as a control. As shown in Fig. 1, the Troygenic producing strain shows a distinct band at the correct height, demonstrating the presence of Troygenics in the culture supernatent.
Amplification of the Application plasmid in the supernatant of an E. coli culture with the Application plasmid (left) and a E. coli culture with the Application and the Assembly plasmid (right).
For the further analysis of our Troygenics, we executed two purification protocols based on PEG/NaCl precipitation, one to purify the Troygenics, the other one to extract the Application plasmid. We verified the correct base sequence of the Application plasmid by nanopore-sequencing the extracted Application plasmid. Additionally, we performed droplet digital PCR to analyse the purity of our Application plasmid. The results depicted in Fig. 2 only show minor contaminations with Assembly plasmid and even less with E. coli DNA.
Relative amounts of Applicaton plasmid, Assembly plasmid and genomic DNA from E. coli in the Application plasmid, purified with the extended protocol from NEB
To demonstrate the production of the Troygenic coat proteins, we performed an SDS-PAGE with the purified Troygenics, which can be seen in Fig. 3. Several bands, which correspond to protein sizes of about 5, 30, 32, 50, 70 and 75 kDa, were visible. The strongest band of about 30 kDa matches the size of pVIII fused to mCherry. The 5 kDa band within the running front could represent the native pVIII. All other bands are bigger than the largest Troygenic coat protein and therefore might correspond to coat proteins which are still connected to each other.
SDS-gel results. Two big bands and five thin bands that indicate the Troygenic proteins are visible.
Finally, we demonstrated the overall correct assembly of our Troygenics by taking an atomic force microscopic picture as shown in Fig. 4.
Atomic force microscopy image of our Troygenic (in the upper middle). Picture taken by Dario Anselmetti.
To further improve the biosafety of our two-plasmid-based Troygenic production system, we aimed to design split selectable markers.

Endocytosis

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Purification of Ligand-mCherry fusion proteins

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Characterization of Ligand-mCherry fusion proteins

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Fluorescence microscopie

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Demonstration of specific endocytosis of S. cerevisiae and A. niger
We constructed the expression plasmids for all Ligand-mCherry fusion proteins successfully and were able to express and purify the proteins. Prior to the endocytosis assays, we characterized all of our Ligand-mCherry fusion proteins. All fusion proteins were analyzed by SDS-PAGE, and showed a band at the right hight respectively. We performed MALDI-TOF analysis of the bands, resulting in the correct identification of all fusion proteins. Additionally, we measured the fluorescence- and absorbance spectra of the fusion proteins, and compared their fluorescence to that of mCherry.
For S. cerevisiae, we performed endocytosis tests with the Ligands Mat, Opy and Flo. The cells were incubated with the fusion protein for more than one hour. We measured the fluorescence intensity in the supernatant in consistent 15 minute intervals. The results shown in Fig 5 display the complete uptake of Mat and Opy by S. cerevisiae within one hour.
Mat_mCherry, Opy_mCherry and mCherry are taken up by S. cerevisiae
S. cerevisiae was incubated in SD media (30 °C, 180 rpm, OD around 0.4, dark) for 1 h with 1 µM mCherry (gray), Mat_mCherry (dark red), Opy_mCherry (dark purple) and Flo_mCherry (purple). A sample was taken every 15 minutes, and the remaining fluorescence in the supernatant measured. (λEx=570 nm, λEm=610 nm, gain calculated from 2.5 µM Texas Red). The results were normalized to the fluorescence intensity at t=0 and the fluorescence intensity of the negative control without cells.

For S. cerevisiae, we additionally performed fluorescence microscopy after we incubated the cells with the fusion proteins for 30 minutes. The microscopic pictures shown in Fig 6 prove the endocytotic uptake of the ligands Mat and Opy, while Flo appeared to form precipitates outside of the cells.
Fluorescence microscopy of S. cerevisiae after incubation with different fusion proteins show specific uptake of them into the cells.
S. cerevisiae (0.35 OD) was resuspended in YPD (60 µL) and incubated (30 min, 30 °C, 450 rpm, dark) with Mat_mCherry (upper right), Opy_mCherry (lower left), Flo_mCherry (lower right) or mCherry (upper left). After washing with PBS half of the cells were visualized using a fluorescence microscope (LSM 700 (Zeiss), filters: Texas Red, transmitted light).

We conducted the same endocytosis assay used for S. cerevisiae with the Pro ligand for A. niger. To test the specifity of the uptake we also tested the uptake of the S. cerevisiae ligand Mat for A. niger. The results are depicted in Fig. 7, showing that A. niger took up Pro, while the fluorescence levels of Mat remained unchanged. This indicates that A. niger is able to execute endocytosis based on specifity.
In conclusion we were able to prove our concept: It is possible to enter selected target cells via receptor-induced endocytosis using cell-sepecific ligands.
The fusion proteins are selectively taken up by the target A. niger.
A. niger was incubated in SD media (30 °C, 180 rpm, dark) for 1 h with 0.5 µM mCherry (gray), Mat_mCherry (dark red) and Pro_mCherry (blue). After 60 minutes a sample was taken and the remaining fluorescence in the supernatant was measured (λEx=570 nm, λEm=610 nm, gain calculated from 2.5 µM Texas Red). The results were normalized to the fluorescence intensity at t=0 and the fluorescence intensity of the negative control without cells.

CeDIS

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Construction of the CeDIS plasmids for S. cerevisiae

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Characterization of Cas13a

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Demonstration of functional CeDIS in S. cerevisiae
We have successfully constructed all CeDIS plasmids with 3 gRNAs for S. cerevisiae. The Cas13a versions Lbu, Lwa and Lsh were assembled with specific promotor-terminator combinations and genomic integration sites for S. cerevisiae. Additionally, we constructed plasmids for the overexpression of all Cas13a versions in E. coli.
We successfully overexpressed and purified Cas13a Lbu and Lsh. The purified proteins were analyzed and identified as correct by performing SDS-PAGE followed by MALDI-TOF analysis of the bands. The in vitro activity analyses of both purified Cas13a variants was determined through the performance of an RNaseAlert assay (Thermo Fisher) with the purified proteins, synthetic sgRNA for RFP and target RNA, which we extracted from an adequate E. coli strain. The results shown in Fig. 8 display clear signs of activity for Lbu and an even higher activity for Lsh. We used Cas13a with guide RNA, but without target RNA, as a negative control to detect offsite cleavage events. Here, we also detected a slight increase in fluorescence, which may be due to offsite activity, but might also be influenced by airborne RNAse ...
In vitro activity of Cas13a Lsh and Cas13a Lbu using the Cas13a activity array based on the RNAse Alert kit. Development of the fluorescence intensity based on the activity of Cas13a Lbu and Lsh over a period of 12 h. For each Cas, the activity was measured when all essential parts for the activation of Cas13a, including the Cas13a protein, gRNA and the target RNA were present. For Lsh, shown in dark purple, and Lbu in dark blue. To account for any offsite events, the activity was also measured without the provision of the target RNA. For Lsh, pictured in red, and Lbu in light blue.

We tested the chosen iducible GALL promotor for Cas13a expression in S. cerevisiae by cloning the gene encoding for sfGFP behind it, and measuring the fluorescence of the yeast culture. The results are shown in Fig. 9. The cells grown on galactose from the beginning showed clear fluorescence after two hours. When grown on raffinose and induced by galactose, the cells showed a fluorescence slightly lower than those grown on galactose for the entirety of the experiment. The cells showed little to no fluorescent activity when grown on glucose, confirming the tight repression of GALL by glucose. Even if the cells are grown on glucose and transferred into galactose medium afterwards, there was almost no fluorescence detected, implying that the GALL promotor was still repressed by glucose.
S. cerevisiae transformed with the Lab application was cultivated in SD media containing raffinose (Raff), glucose (Glu) or galactose (Gal) (30 °C, 180 rpm, OD). Samples marked with ind. were induced with 2% galactose at an OD of 0.4. The measurement took place 2 h after induction. Three samples were taken of each culture and the fluorescence was measured using the TECAN infinite M200 (λEx=570 nm, λEm=610 nm, gain calculated from 2.5 µM fluorescein). The results were normalized to the max fluorescence intensity of cells continuously grown on raffinose

For the CeDIS tests with Cas13a Lsh and Cas13a Lbu, we used S. cerevisiae strains transformed with the CeDIS plasmids containing three sgRNAs. We cultivated S. cerevisiae on raffinose as C-source and induced the Cas13a expression by addition of galactose in the exponential growth phase. The results depicted in Fig. 10 and 11 clearly show that the cells reach a premature stationary phase. This indicates that the growth of S. cerevisiae is halted. Over the course of about 10 hours, there was no significant increase in the OD of the induced cells carrying the CeDIS. However, the uninduced samples containing Cas13a also show a greatly reduced growth. This deviation from S. cerevisiae without Cas13a can be assigned to the choice of carbon source for this growth experiment. As previously shown in Fig. 9, the GALL promotor is activated by raffinose almost as strongly as it is activated by galactose. Therefore, Cas13a is produced at a low rate, even in the uninduced state, which leads to a decrease in growth.
Growth experiment of S.cerevisea transformed with pRS304_Lsh to assess the functionality of the CeDIS All yeasts were cultivated on raffinose containing SD medium in 250mL shaker flasks at a temperature of 30 degree celsius at 180 rpm. As a control the wild type S.cerevisea was grown on raffinose and at the point of induction inhibited with 4% glucose(red), as well as treated as if it would be induced with 2% galactose (dark purple). S. cerevisea transformed with pRS304_Lsh was grown on selective SD medium with raffinose as a carbon source and at point of induction inhibited by addition of 4% glucose(dark blue). Furthermore an galactose induced culture containing pRS304_Lsh (light blue) was measured

Growth experiment of S.cerevisea transformed with pRS304_Lwa to assess the functionality of the CeDIS All yeasts were cultivated on raffinose containing SD medium in 250mL shaker flasks at a temperature of 30 degree celsius at 180 rpm. As a control the wild type S.cerevisea was grown on raffinose and at the point of induction inhibited with 4% glucose(red), as well as treated as if it would be induced with 2% galactose (dark purple). S. cerevisea transformed with pRS304_Lwa was grown on selective SD medium with raffinose as a carbon source, and at point of induction inhibited by addition of 4% glucose(dark blue). Furthermore an galactose induced culture containing pRS304_Lwa (light blue) was measured

In conclusion, we were able to show that the CeDIS based on Cas13a Lsh or Cas13a Lbu with three sgRNAs works for S. cerevisiae. Adittionaly, we demonstrated that Cas13a Lsh and Cas13a Lbu are active and working target specific in vitro.

Proof of concept

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Construction of the Application plasmid for S. cerevisiae

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Transformation of S. cerevisiae

We have successfully assembled two Application plasmids for the transformation of S. cerevisiae. Both encode the shortened PIII protein and the lab application basic insert for S. cerevisiae. BBa_K2926093 is encoding a PVIII mCherry Mat fusion protein and BBa_K2926022 encodes a PVIII mCherry Opy fusion protein. The correct nucleotide sequences were confirmed by Sanger sequencing. After the co-transformation of the Assembly plasmid and one of the Application plasmids each, we produced and purified our Troygenics. Since our Troygenics present mCherry on their coat, we measured the fluorescence of the purified Troygenic which was produced with the Assembly plasmid , to prove their correct assembly. As depicted in Fig. 12, we observed a peak at 500 to 650 nm on the emission spectrum, which matches the typical emission of mCherry. The slight peak-shift is most likely due to the fact that the mCherry is fused to the Troygenic. The spectrum indicates that we fused the ligands which are required for target specific encocytosis with mCherry and pVIII correctly. Additionally, it demonstrates that mCherry is represented on the surface of our Troygenics as planned.
Fluorescence of Troygenics built with BBa_K2926093 compared to mCherry.

Subsequently, we incubated 1.5mL S. cerevisiae over-night-culture with 50 μL of purified Troygenics for 30 minutes, and afterwards plated them on selection plates. As a control we incubated cells with 2 ng circular Assembly plasmid, which equals the amount of DNA usually used for a transformation with S. cerevisiae. We performed a colony PCR on the growing clones with primers specific for the Assembly plasmid. The results shown in Fig. 13 depict distinct bands at the appropriate height for the colonies incubated with purified Troygenics, while there are no visible bands for the colonies that were incubated only with Application only.
Colony PCR on S. cerevisiae treated with Troygencis built with BBa_K2926093.

Hardware

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Construction of a low-budget DIY starter set for microfluidics

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Construction of microfluidic chips for cultivation

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On chip cultivation of S. cerevisiae and A. niger
We designed and constructed two microfluidic chips for the cultivation of S. cerevisiae and A. niger. The chips were manufactured using a 3D-printed waver that was optimized several times during the period of our project. These chips enabled us to observe our eucaryotic target cells in single-cell-resoulution. Quick-motion recordings of on-chip cultivations of S. cerevisiae and A. niger are shown in Fig. 14 and 15.
Quick-motion on-chip cultivation of S. cerevisiae (real-time cultivation of about 60 minutes)
Quick-motion on-chip cultivation of A. niger (real-time cultivation of 34 hours).
To give other iGEM teams access to the possibilities of micorfluidic systems, we have also designed and assembled a microfluidic lab starter kit. It contains a PDMS-stirrer, a UV chamber, an oven, and a syringe pump: Almost all tool required for running a micofluidic lab, including chip construction, are included. All parts of our microfluidic lab starter kit can be 3D-printed. The blueprints as well as short and easily understandable construction manuals are available on our download and hardware pages. Our low-cost microfluidic lab starter kit enables other iGEM teams to build their own microfluidic lab for less than 50$.