What work we've done:

Verified the concept of our design for three modules respectively.

Verified the activity of EpCAM.

Verified the successful combination between SYL3C aptamer and EpCAM.

Verified that SYL3C aptamer could combine with 10nt complementary chain successfully.

Verified the feasibility of “competition method” successfully.

Verified that As Cas12a could be activated.

Verified the trans-cleavage activity.

Transferred molecular signal to fluorescence signal with DNase Alert.

Verified the transfer from protein signal to the fluorescent one successfully

The work hasn’t finished yet:

Quantitative analysis of the effect of "competition method" and "As Cas12a system" respectively

Verified the detection limit of "competition method", "As Cas12a system", and then our whole system

Other designs to transfer signals for reporting devices

Activity of EpCAM (Flow cytometry)

We got EpCAM directly from ACROBiosystems. In order to prove that the protein was still bio-active, we chose antibody of EpCAM with fluorescence and flow cytometry to measure it. Furnished by the research group laboratory, the fluorescence antibody was proved effective through previous experiments.

We bought EpCAM with His-tag tag. It’s widely known that His-tag could chelate with nickel ions, so we incubated EpCAM protein with nickel beads to bind them together. Adding the antibody and then incubating, we measured them with flow cytometry. At the same time, we started two other control group experiments, with the result shown below. Get more information of our experiment, click here.

Figure 1: the result of detecting the binding of EpCAM with fluorescent antibody, with flow cytometry.
a) Black line: pure nickel beads;
b) Blue line: nickel beads + fluorescent antibody (measure the non-specific adsorption between beads and antibody);
c) Red line: nickel beads - EpCAM complex + fluorescence antibody.

From the graph and data above, we could find out that the non-specific adsorption between nickel beads and fluorescent antibody does exist, but in the presence of EpCAM, the fluorescence intensity increased seven times compared with the original one, so we could prove that our bio-active EpCAM protein successfully combined with fluorescent antibody.

Combination between SYL3C and EpCAM (fluorescence polarization)

Although EpCAM and aptamer SYL3C are directly quoted in the literature ^[1], we believed that we still needed to verify this step and that we could ensure the validity of our material.

We struggled with this step for a long period, and we suspected that our failure was caused by impure materials. We used 30KDa ultrafiltration tube - through which unbound fluorescent aptamers could pass, but the aptamer combined with protein could not. We also used flow cytometry to directly detect whether there were binding fluorescent aptamers on EpCAM, without satisfying results in the end.

Fortunately, we successfully validated this step with the fluorescence polarization method after replacing materials, with the proposal from Prof. Zhi Zhu. We incubated two groups, namely pure fluorescence aptamers as well as fluorescent aptamers with EpCAM without light at the same time, then adding binding buffer to the total volume of 200μL. After incubation, we added 3mL binding buffer for fluorescence polarization. Get more information of our experiment, click here.

Figure 2.1: polarizing fluorescence intensity of fluorescence aptamer.

Figure 2.2: polarizing fluorescence intensity of fluorescent aptamers with EpCAM, after incubation.

According to the principle of fluorescence polarization, the FP value would depend directly on the effective molecular size of the fluorescent group at fixed temperature and viscosity of solution. For small molecules (fluorescent aptamers) with fast Brownian rotation in solution, the FP value was lower; while for larger molecules (fluorescent aptamers combined with EpCAM), the FP value was higher. ^[2]

The FP value could be calculated by the following formula:

Below are four maximum fluorescence values for calculating FP values:

Later, we could calculate whether the FP value of fluorescent aptamers increased with EpCAM incubation according to the principle. If the FP value increased, it showed that fluorescent aptamers did bind to EpCAM. We calculated the difference herein:

With an increase of 0.142, the difference was rather obvious, which demonstrated that the fluorescent aptamer did combine with EpCAM.

Combination between SYL3C and the “complementary chain” (DNA agarose gel electrophoresis)

We've been struggling for some time due to one problem about how to prove that our aptamers and the complementary chains were combined, which would be paramount for our following experiments.

We tried SYBR Green I, a fluorescent dye that could be embedded in a double helix of DNA and thus significantly enhance fluorescence intensity, since it could distinguish ssDNA from dsDNA obviously. However, there's no significant difference according to our results. We speculated that the sequence might be not long enough, coupled with the formation of secondary structure of aptamers, leading to no difference in fluorescence intensity.

Finally, we exploited the conventional gel electrophoresis method to prove this block, and then gel electrophoresis was performed immediately after grouping and denaturing annealing. Because the sequence was too short, we did not have the appropriate DNA marker to use. Get more information of our experiment, click here.

Figure 3: gel image of aptamer combined with complementary chain.
a) W1: aptamer;
b) W2: 24nt complementary chain with a 9nt complementary part;
c) W3: 24nt complementary chain with a 10nt complementary part;
d) W4: Aptamer+ 24nt complementary chain with a 9nt complementary part;
e) W5: Aptamer+ 24nt complementary chain with a 10nt complementary part;

We could find that there were three distinct bands between the aptamer + complementary chains with a 10nt complementary part (W5), while 9nT did not. The lane 1 and 2 acted as markers, so we could clearly identify the positions of the aptamers and complementary chains in the three bands. The last band, obviously, was the result of an increase in molecular weight, which meant that the aptamer and the complementary chain combined together successfully.

Therefore, we have proved that the aptamer could combine with its complementary chain successfully.

The competition (Spectrofluorometer)

We used the competition method to transfer molecular signal, because we considered that the competition method was best operated automatically later, so that users did not need to have a deep understanding toward technical basis. Therefore, it's quite important for us to verify that EpCAM could compete successfully, from a theoretical prospective to support our hardware and the activation of Cas12a as well.

Below are the exact procedures of our experimental group. First, we successfully hatched the streptavidin beads, biotin-modified aptamers and fluorescent complementary chains to form the complexes, then fixed the beads with magnets and discarded the supernatant, which would still contain fluorescent complementary chains that did not form complexes. After that, we incubated the complex with EpCAM to achieve the process of competition.

Additionally, considering the non-specific adsorption, we set up several control groups, and the variables amongst them were replaced by buffer. After competition, we fixed the magnetic beads with magnet, then extracted the supernatant and added 3mL incubation buffer in tandem. Below is the plot about the fluorescence intensity detected in different groups. Get more information of our experiment, click here.

Figure 4: fluorescence intensity images of competition results
a) Black line: incubation buffer;
b) Red line: magnetic beads - aptamer + incubation buffer;
c) Blue line: magnetic beads - aptamer - complementary chain complex + incubation buffer;
d) Green line: magnetic beads - aptamer - complementary chain complex + EpCAM + incubate buffer.

Comparing the four groups, we can conclude that the non-specific adsorption of complementary chains on the magnetic beads does exist, and the magnetic bead-aptamer complex, like the buffer solution, had almost no fluorescence. Significantly, the fluorescence intensity of the competition group with EpCAM was about 2.5 times larger than that of the non-specific adsorption group, which demonstrated that the fluorescence complementary chain of the competition group with EpCAM was successful, and so was our competition method.

Trans-cleavage activity of Cas12a (Microplate Reader)

It's reported that cas12a could be activated by ssDNA and has trans-cleavage activity this year, so we hope to use this function in our system to amplify the signal. ^[3]

We operated the experiment according to the instructions from IDT. We incubated the cas12a protein with crRNA to form a complex and then added our ssDNA to be recognized. Then, we used the commercial DNase Alert Kit to explore its trans-cleavage activity. We drawed the fluorescence curve with time using a microplate reader, and measured the fluorescence from 30 minutes to 110 minutes

In order to control variables, we also set up a negative (DNase Alert) and a positive (DNase I + DNase Alert) control group. Get more information of our experiment, click here.

Figure 5: ffluorescence time curve image of activation effect of cas12a
a) Black line: negative group, DNase Alert only;
b) Red line: positive group, DNase I + DNase Alert;
c) Blue line: experimental group, activated cas12a+DNase Alert;

We fitted three sets of data points with Gaussian fitting curve in Figure 5. From the fitting results, we could see that the negative group and the positive group were declining, while the cas12a proteome increased at first and then decreased. This is in line with our expectations, and the reason for the decline was, we suspected, the quenching caused by continuous measurements and the weakening of the fluorescence itself as the experiment proceeds. However, it's enough to prove that cas12a could meet the expected result of amplifying signals.

Signal transformation by DNase Alert (Spectrofluorometer)

Although we have used DNase alert to detect Cas12a, as the last of the three modules we designed, we needed to prove that DNase alert was indeed cut and would emit fluorescence to our expectations, so that it could be used to transfer our signals.

Here, we chose DNase I to verify directly. In the presence or absence of DNase I, we measured fluorescence intensity in the DNase alert tube. Considering the experiences we got previously that long-term measurement in the microplate reader would lead to reduction in fluorescence intensity, resulting in experimental error, we chose spectrophotometer instead.

We set up the experimental group (with DNase I) and the control group (without DNase I), with the time gradient of 3, 15, 30 minutes. All groups started at the same time. We didn’t operate continuous measurement, to avoid excitation that may cause partial fluorescence quenching. After incubation, 3mL TE buffer was added, and then detect the fluorescence. Get more information of our experiment, click here.

Figure 6: fluorescence intensity curve in 3rd minutes, with or without DNase I

Figure 7: fluorescence intensity curve in 15th minutes, with or without DNase I

Figure 8: fluorescence intensity curve in 30th minutes, with or without DNase I

From the Figure 6-8, we could find that cutting efficiency of DNase I was very high, almost all of the DNase Alert substrate has been cut in 3 minutes, fluorescence in 15 and 30 minutes and 3 minutes almost keep the same level. As we expected, DNase alert could indeed be cleaved and then re-released as a fluorescent group. In other words, DNase alert could transfer the molecular signal to the fluorescent signal actually.

Integrated experiment

After making each part work effectively, we accomplished the integrated experiment finally, transferring the protein signal to fluorescent one.

Similarly, following procedures mentioned in the "competition method" part, we successfully hatched the streptavidin beads, biotin-modified aptamers and fluorescent complementary chains to form the complexes, then fixed the beads with magnets and discarded the supernatant, which would still contain fluorescent complementary chains that did not form complexes. After that, we incubated the complex with EpCAM to achieve the process of competition. After competition, we fixed the magnetic beads with magnet, adding the supernatant extracted into Cas12a-crRNA complex incubated previously to activate Cas12a protein. Finally, we added it into DNase Alert, supplementing 3mL TE buffer after 30 minutes to measure the fluorescent intensity. Below is the corresponding plot we've obtained, indicating that we made the integrated experiment successfully.

Figure 9: Integrated experiment

References

[1] Yanling Song, Zhi Zhu, Yuan An, Weiting Zhang, Huimin Zhang, Dan Liu, Chundong Yu, Wei Duan, Chaoyong James Yang, Selection of DNA Aptamers against Epithelial Cell Adhesion Molecule for Cancer Cell Imaging and Circulating Tumor Cell Capture, Anal Chem, 2013, 85, 4141-4149.
[2] DS Smith, SA Eremin, Fluorescence polarization immunoassays and related methods for simple, high-throughput screening of small molecules, Analytical and bioanalytical chemistry, 2008, 391 (5) :1499.
[3] Chen JS, Ma E, Harrington LB, Da Costa M, Tian X, Palefsky JM, Doudna JA, CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity, Science, 2018, 360 (6387) : seaar6245

Background

Outer-membrane vesicles (OMVs) are lipid vesicles commonly produced by Gram-negative bacteria, which are filled with periplasmic content and are 20-250 nm in diameters (Figure 1). The production of OMVs allows bacteria to interact with their environment, and OMVs have been found to mediate diverse functions, including promoting pathogenesis, and enabling bacterial delivery of nucleic acids and proteins ^{[3] [4]}. A recent paper by Kojima R et al. 2018, demonstrated an EXOtic device that can produce exosomes with specific nucleic acids cargo ^[1] (Figure 2). We were impressed by the amazing OMVs and EXOtic device and came up with an idea to design a cell-free system to enable specific siRNA to be encapsulated into OMVs for cancer treatment.

Figure 2. The cell envelope of Gram-negative bacteria consists of two membranes, the outer membrane and the cytoplasmic membrane. Envelope stability comes from various crosslinks including the non-covalent interactions between the PG and the porin outer-membrane protein A (OmpA).

Figure 3. Schematic illustration of the EXOtic devices. Exosomes are nanoscale extracellular lipid bilayer vesicles of endocytic origin, and they are secreted by nearly all cell types in physiological and pathological conditions. Exosomes containing the RNA packaging device (CD63-L7Ae) and mRNA (e.g., nluc-C/Dbox) can efficiently deliver specific nucleic acids.

Abstract

Not only eukaryotes but also prokaryotes can produce nanoscale bubbles to fulfill diverse functions, such as cellular communication, surface modifications and the elimination of undesired components. Additionally, because of this functional versatility, OMVs have been explored as a platform for bioengineering applications. This year, we XMU-China decide to utilize OMVs as a cell-free platform to deliver our nucleic acids agents to facilitate therapeutic targeting of oncogenic KRAS in pancreatic cancer ^{[5] [6]}.

Figure 3. We utilize a split protein SpyTag/SpyCatcher (ST/SC) bioconjugation system to create a synthetic linkage between protein OmpA and archaeal ribosomal protein L7Ae. We fuse SpyTag with OmpA at its C-termini and N-termini respectively.^[2]

Figure 5. After the induction of IPTG and Arabinose, we can get L7Ae-SpyCatcher and siRNA-C/Dbox. Archaeal ribosomal protein L7Ae owns the ability to bind with C/Dbox RNA structure.

Figure 6. With the interaction between SpyTag and SpyCatcher, and the ability of L7Ae to be bind with C/Dbox, we can produce customizable and cell-free OMVs containing specific siRNA to traget for oncogenic gene.

References

[1] Kojima R, Bojar D, Rizzi G, et al. Designer exosomes produced by implanted cells intracerebrally deliver therapeutic cargo for Parkinson’s disease treatment[J]. Nature Communications. 2018, 9(1):1305.
[2] Alves N J, Turner K B, Medintz I L, et al. Protecting enzymatic function through directed packaging into bacterial outer membrane vesicles: [J]. Scientific Reports, 2016, 6:24866.
[3] Schwechheimer C, Kuehn M J. Outer-membrane vesicles from Gram-negative bacteria: biogenesis and functions. [J]. Nature Reviews Microbiology, 2015, 13(10):605-19.
[4] Vanaja S K, Russo A J, Behl B, et al. Bacterial Outer Membrane Vesicles Mediate Cytosolic Localization of LPS and Caspase-11 Activation. [J]. Cell, 2016, 165(5):1106-1119.
[5] Kamerkar S, Lebleu V S, Sugimoto H, et al. Exosomes facilitate therapeutic targeting of oncogenic KRAS in pancreatic cancer[J]. Nature, 2017, 546(7659):498-503.
[6] https://en.wikipedia.org/wiki/Pancreatic_cancer

Background

Tardigrades are able to tolerate almost complete dehydration by reversibly switching to an ametabolic state. This ability is called anhydrobiosis.^[1]Tardigrade-specific intrinsically disordered proteins (TDPs) are essential for desiccation olerance.^[2]2012, Takekazu Kunieda and his team identified five abundant heat-soluble proteins in the tardigrades, which can prevent protein-aggregation in dehydrated conditions in other anhydrobiotic organisms.^[1]

Figure 7. Stage Photo of Tardigrades in Ant-Man 2. ^[4]

In 2017, Thomas C. Boothby and his team segregated three TDP proteins in the water bears and explored their mechanism of action^[3]. This is a schematic diagram of the mechanism they have done so far. At the same time, one of the 2017 iGEM teams TUDelft, attempted to preserve the Cas13a protein using the TDP proteins, and they also tried to preserve the bacteria with the TDP proteins and obtained amazing outcome. In our project, we are going to use TDPs to help preserve the protein Cas12a and OMVs.

Abstract

We have carried out research on TDP proteins this year. On the one hand, we plan to preserve the Cas12a required for protein detection and OMVs required for treatment with TDPs. On the other hand, as the wiki says, TDP is a new biological activity protector with great potential. So we are going to use TDP proteins to simplify existing methods of preserving proteins and bacteria. There are two novel protein families with distinct subcellular localizations named Cytoplasmic Abundant Heat Soluble (CAHS) and Secretory Abundant Heat Soluble (SAHS) protein families, according to their localization. In our project, SAHS1 was used to preserve the proteins and CAHS1 was used for the preservation of the bacteria.

Figure 8. The Expression of TDPs When The Tardigrades Suffer Form Fast Drying and Slow Drying.(Thomas C. Boothby et al. 2017).

References

[1]. Yamaguchi A. Two Novel Heat-Soluble Protein Families Abundantly Expressed in an Anhydrobiotic Tardigrade. PLoS ONE, 2012;7(8):e44209.
[2]. Boothby TC. Tardigrades Use Intrinsically Disordered Proteins to Survive Desiccation. Mol Cell. 2017 Mar16;65(6):975-984.e5.
[3]. Boothby TC. Intrinsically Disordered Proteins and Desiccation Tolerance: Elucidating Functional and Mechanistic Underpinnings of Anhydrobiosis. Bioessays. 2017 Nov;39(11):1700119.
[4]. https://www.marvel.com/