iGEM 2019 — Team XJTLU-CHINA

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# Team:XJTLU-CHINA/Notebook

Xi'an Jiaotong-Liverpool University

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# Team:XJTLU-CHINA/Experiments

Xi'an Jiaotong-Liverpool University

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* [Literature Research](#Title1)
* [Experiment Design](#Title2)
* [Notebook](#Title3)
* [Protocols](#Title4)
* [References](#Title5)
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### Literature Research

#### EAAT2

Glutamate, the primary excitatory amino acid neurotransmitter, in the central nervous system (CNS) is also a powerful neurotoxin that may result in the nerve cells’ death. It must be taken up by the synaptic cleft when participating in the signaling process from glutamatergic nerve endings to various types of glutamatergic receptors (Kanai and Hediger, 2003). Otherwise, a large number of detrimental consequences such as calcium homeostasis dysfunction, increased nitric oxide (NO) production, activation of proteases, an increase in cytotoxic transcription factors and increased free radicals will be caused after glutamate receptors receive prolonged stimulation (Wang and Qin, 2010). Therefore, many neurological disorders such as epilepsy, stroke and neurodegenerative diseases, and dysfunctional glutamate transporters have a strong association with increasing glutamate levels, which are likely to initiate the brain injury (Kim et al., 2011).

![](https://static.igem.org/mediawiki/2019/9/9b/T--XJTLU-CHINA--E1.jpg)

 Figure 1: Glutamate is an important neurotransmitter. However, high concentration of glutamate could result in neurovirulence and cause neurodegeneration.

The glutamate concentration in the synaptic cleft and its post-synaptic activity of glutamate receptors are highly related to the dynamic equilibrium between the release and clearance of glutamate (Kanai and Hediger, 2003). In order to terminate synaptic transmission, glutamate transporters control glutamate within a low level by immediately removing it from the extracellular space after releasing caused by an action potential (Beart and O’Shea, 2007).

![](https://static.igem.org/mediawiki/2019/7/75/T--XJTLU-CHINA--E2.png)

 Figure 2: Picture shows how glutamate is released and removed in synaptic clefts. Transporter proteins are showed in green.

The high-affinity sodium-dependent transporters are regarded as the predominant mechanism in the brain for maintaining the stable concentration glutamate by absorbing this excitotoxic amino acid. The EAATs located on the membrane of astroglia or in glia which closely resemble ion channels are known to be the notable transporters to treat the prolonged exciting problem (Kim et al., 2011).

![](https://static.igem.org/mediawiki/2019/6/62/T--XJTLU-CHINA--E3.png)

Figure 3: Astroglia membrane anchors EAAT proteins that could efficiently transmit glutamate from synaptic clefts.

The EAATs are symporters which means that the transporter protein transport a substrate by the co- and counter-transport of ions (Robinson,1998). The initiation of transport cycle is triggered by binding of a substrate molecule, three sodium ions and a proton to form an outward-facing EAAT conformation. Afterwards, the substrates, sodium ions and protons and are released into the cytoplasm of the cell, thereby resulting in an inward-facing conformation formed. Subsequently, after the counter-transport of a potassium, the new substrate molecule become accessible again and the transporter will also return to its outward-facing conformation (Levy et al., 1998).

Since the activities of EAAt2 are regulated both transcriptionally and post-transcriptionally, up-regulation of EAAT2 are likely to be observed at both transcriptional and translational level.

![](https://static.igem.org/mediawiki/2019/3/31/T--XJTLU-CHINA--E4.png)

Figure 4: Intracellular regulation of expression of EAAT2 is controlled in both transcriptional level and translational level. Post-transcriptional modification and post-translational modifications are important in EAAT2 expression.

For transcriptional example:
NF-kB is primarily a transcriptional activator, which can also act as a transcriptional repressor in some cases. It mediate the expression of many essential molecules such as epithelial cell adhesion molecule (EpCAM) and cytochrome P-4501A1. According to the study of Sitcheran et al. (2005), NF-kB directly binds to the EAAT2 promoter, and regulates its transcription. The studies with pharmacological and genetic inhibitors suggest that the EAAT2 expression depend on the signaling pathways through PI3K and NFkB (Kim et al., 2011).

For post-transcriptional example：
A study have previously found that a SUMO1- conjugated C-terminal fragment of EAAT2 (termed CTESUMO1) accumulates in ALS (Foran et al., 2014). Small ubiquitin modifier 1 (SUMO1) is a posttranslational modifier protein, which attaches to lysine residues via an isopeptide bond. (Foran et al., 2014). EAAT2s modified with SUMO1 have been changed in localization, trafficking, and half-life due to this ubiquitination modification. Since EAAT2 with this modification can no longer be maintained at the cell membrane, it would lost the transport fuction immediately and be degenerated in a short period (Foran et al., 2014).

![](https://static.igem.org/mediawiki/2019/7/7e/T--XJTLU-CHINA--E5.png)

 Figure 5: Astrocytic dysfunction eventually cause glutamate increase and results in neuron death.

According to previous research, dysfunction or decreased expression of EAAT2 associated with impaired glutamate uptake has been implicated in many pathogenesis of diseases. In extracellular fluid, down-regulation of EAAT2 followed by accumulation of glutamate leading to neuronal death have been recorded in diverse aetiology, including Alzheimer’s disease, several types of dementia, etc (Liu et al., 2017)

#### Cx43

Connexin 43 (Cx) proteins can be assembled into a six-protein oligomer called a connexon. Connexons are inserted into the plasma membrane (Falk et al., 2016). During cell-cell contact, the hemichannels can dock head-on with the hemichannels from another cell, which is exosomes in our project. These connexons can then cluster to form a gap junction (GJ) plaque. The plaque can be used for rapid material exchange between exosomes and target cells (Falk et al., 2016). Once the plaque is no longer needed for cell-cell communication, the central portion of the plaque or the entire plaque would be internalized to form an annular gap junction. The annular gap junction may then be degraded via lysosomal degradation (Falk et al., 2016). The free connexin proteins in cytoplasmic membranes that are released during vesicle degradation may then be recycled back to the cell surface to participate in the formation of new, or the addition to existing gap junction plaques (Falk et al., 2016).

![](https://static.igem.org/mediawiki/2019/c/c5/T--XJTLU-CHINA--E6.png)

 Figure 6: (Solan and Lampe, 2018). Flowchart of connexon mediated membrane fusion. Connexons on cell membrane could initialize membrane fusion process between two membranes, therefore enhance cargo delivery of exosome.

The consequences of this rapid turnover showing that these proteins generate subcellular structures that are in a constant state of flux. In a normal situation, the size and longevity of individual gap junctions can be balanced through the accretion of “new” protein and internalization of the oldest protein (Solan and Lampe, 2018).

![](https://static.igem.org/mediawiki/2019/3/3f/T--XJTLU-CHINA--E7.png)

 Figure 7: (Falk et al., 2016). The detailed mechanism of connexon mediated membrane fusion. Phosphorylation of Ser 368 is important for internalization.

The phosphorylation of Ser 368 is required for conformational alteration of the Connexon, which is also likely to play an essential role in internalization. When gap junction channels aged (permanently closed) and move inward towards plaque centres, the phosphorylation of S368 allows ZO-1-bound to be transited into ZO-1-unbound gap junction channels. The gap junction channels would be closed and be internalized (Falk et al., 2016). However, in our project, we used the S368A mutant of Cx43. Without the serine in 368 positions, the ZO-1 would be kept binding to gap junction channels, and the gap junction plaques can not be internalized. The gap junction plaques would be kept at the efficient transition state (Falk et al., 2016).

#### IVT delivery system

Injection of in vitro transcribed (IVT) mRNA could be used to express encoded proteins in the injected tissue in living organisms for delivering genetic information. Structural assembling of natural mRNA could be engineered to synthetic mRNA for transient expression of proteins in the transfected cells (Sahin et al., 2014). Contrast to traditional DNA therapies that requires access of exogenous DNA into the nucleus for RNA transcription, IVT mRNA could deliver its function by only reaching the cytoplasm and translating the mRNA without entering nucleus. Therefore it is free of risks of insertion mutagenesis into cell genome and induce potential ethical problems. Traditional therapeutic DNA treatments also depends it functionality on nuclear envelope breakdown during mitosis, which could be a limit of functional efficiency (Sahin et al., 2014).

IVT mRNA also have advantages over most pharmaceutical therapies due to its transient activity and fast degradation in vivo. Synthetic mRNA is also cost less to produce, and could functionality could be easily modified with knowledge of related genetic materials. In different clinical areas, IVT mRNA is either at preclinical stages or has reached Phase III clinical testing (in cancer immunotherapy) (Heiser et al., 2002, Morse et al., 2002). When used for ex vivo cell therapeutic applications, IVT mRNA therapy would be challenged by industrialization hurdles for cell therapies, but in the case of in vivo usae, IVT mRNA has the advantage of low cost, easily manufactural and free of many mutagenesis risks (Sahin et al., 2014).

### Experiment design

#### EAAT2-Functional Characterization

**1. Transfection of EAAT2-C/D Box-pcDNA3.1(+)**

To characterize our part designed for coding expressing EAAT2, we transfected the part BBa\_k3030002 on pcDNA3.1(+) using engreen the transfecting reagent Engreen to P4 of N2a cells, which have multiple branches that mimic appear in normal neurons and glial cells. All of the Neuro-2a cells were incubated in 5% FBS-added DMEM medium with anti-anti in a 24-well plate until the cells were grown to approximately 60% confluence. In this part of experiment, the empty plasmid pcDNA3.1(+) was used as the negative control plasmid, and transfected into another 12 wells, the distribution of the samples in each well is displayed as below:

![](https://static.igem.org/mediawiki/2019/9/93/T--XJTLU-CHINA--E8.jpg)

Our operation in this experiment follows the protocol for 24-well engreen transfection: [Protocol link.](https://static.igem.org/mediawiki/2019/7/73/T--XJTLU-CHINA--Transfection-Engreen.pdf)

**2. Cell Nurturing: Inflammation-inducing Condition**

12 hours after the transfection of BBa\_k3030002 and BBa\_k3030004, the medium of N2a cells were was changed to the normal exosome-free DMEM medium.

To induce inflammation in the N2a cells, we changed the medium to phenol-free RPMI1640 medium with 5% exosome-free FBS, and additional glutamate were was added to ensure that the concentration of glutamate is was 80 uM.

The time interval of changing DMEM medium to high glutamate -containinged medium is was controlled to three hours, and for both of the EAAT2-transfected and pcDNA-transfected N2a cells, there are were four groups of cells to be manipulated, and each group has was present in three wells. The distribution of sample in each well is displayed as below:

![](https://static.igem.org/mediawiki/2019/3/3c/T--XJTLU-CHINA--E9.png)

**3. WST-1**

To test the cytotoxicity induced by glutamate to in N2a cells, we used Beyotime WST-1 Cell Proliferation and Cytotoxicity Assay Kit. In each well, we added 20 ul WST-1 in 200 ul medium in each well of the 24-well plate. After incubating the WST-1-added cells in 37 ℃ for 2 hours, we measured the absorbance at 450 nm, and 620 nm was used as reference wavelength in a 96-well plate.

Our operation in this experiment follows the protocol for WST-1 Test: [Protocol link.](https://static.igem.org/mediawiki/2019/b/bc/T--XJTLU-CHINA--WST-1.pdf)

**4. Glutamate Assay**

To detect the exact glutamate concentration in the cell medium that mimics the cerebrospinal fluid, we used the Enzychrom Glutamate Assay Kit. The cell medium from each well was extracted, while 20 ul in each sample was used in the glutamate assay. The measurement of absorbance at 565 nm was conducted twice respectively: at the moment immediately after adding the working reagent solution and after a 30-minute incubation at room temperature.

Our operation in this experiment follows the protocol for Glutamate Assay: [Protocol link](https://static.igem.org/mediawiki/2019/3/3e/T--XJTLU-CHINA--GlutamateAssay.pdf).

#### EAAT2-Exosome Delivery

**1. Cx43 Transfection**

To ensure the exosome to specifically combine and deliver cargo to neurons and glial cells containing Cx43 on membrane surface, we transfected Cx43 coding sequence on pcDNA3.1 (+) into the P3 HEK293T cells (exosome donor) and P4 N2a cells (exosome receptor) using engreen transfection kit. Both of the HEK293T and N2a cells were respectively incubated in DMEM medium with 5% exosome-free FBS and 1% anti-anti in two T25 flasks. In this part of experiment, the empty pcDNA3.1(+) was used as the negative control plasmid, and transfected in the other two T25 flasks which share the same incubating condition with the Cx43-trasfected cells.

Our operation in this experiment follows the protocol for T25 plate engreen transfection:
[Protocol link.](https://static.igem.org/mediawiki/2019/7/73/T--XJTLU-CHINA--Transfection-Engreen.pdf)

**2. Ultracentrifugation: Exosome Isolation**

12 hours after we finished the transfection of Cx43 and pcDNA3.1(+) in two HEK293T flasks, the medium was changed to exosome-free DMEM medium for cell culture. After 24 hours, the cell medium was extracted for ultracentrifugation. The extracted exosome pellets were resuspended in 50 ul cold sterile PBS and well-stored in a -80 ℃ freezer.

Our operation in this experiment follows the protocol for ultracentrifugation:
[Protocol link.](https://static.igem.org/mediawiki/2019/3/3c/T--XJTLU-CHINA--Exosome_Extraction-Ultracentrifugation.pdf)

**3. Endo-free Miniprep of EAAT2-C/D Box-pcDNA3.1(+)**

Before transfecting the mRNA into the exosomes (and the N2a cells), we need to get the endotoxin-free plasmids which can be transcribed to mRNA. Thus, we used Axygen Endo-free Plasmid Kit for DNA isolation of DH5a that transformed with part BBa\_k3030002 and with BBa\_k3030004. The isolated plasmid was then stored in a -20 ℃ freezer.

Our operation in this experiment follows the protocol for Endo-free Miniprep:
[Protocol link.](https://static.igem.org/mediawiki/2019/e/ed/T--XJTLU-CHINA--EndofreeMiniprep.pdf)

**4. In Vitro Transcription**

We used MAXIscript Kit from Ambion for in vitro transcription of our isolated EAAT2-C/D Box-pcDNA3.1(+) and empty pcDNA3.1(+). To prevent the premature termination, we extend the reaction time to 2 hours, decline the reaction temperature to 15 ℃, and rise the concentration of each NTP to 500 M. After accomplishing the IVT, we added 1 ul TURBO DNase each sample to remove the template DNA in each sample, and incubated at 37 ℃for 15 minutes.

Our operation in this experiment follows the protocol for In Vitro Transcription:
[Protocol link.](https://static.igem.org/mediawiki/2019/b/bb/T--XJTLU-CHINA--InVitroTranscription.pdf)

**5. Polyadenylation**

In order to protect the naked mRNA transcribed, we used Poly[A] Enzyme from Thermo Fisher Advanced miRNA Assay Kit to add poly-A tails on the 3’ ends.

Our operation in this experiment follows the protocol for Polyadenylation:
[Protocol link.](https://static.igem.org/mediawiki/2019/4/4d/T--XJTLU-CHINA--Polyadenylation.pdf)

**6. RT-PCR**

To calculate the approximate length of mRNA in vitro transcribed, we used universal RT primers and RT-PCR master mix in Taqman Advanced miRNA cDNA Synthesis Kit to produce cDNA of the transcribed mRNA. Before conducting the reverse transcription, the sample were incubated at 85 ℃ for 3 minutes to inactivate the TURBO DNase added in step 4. For each reaction, the reagent mix is added as followed instruction (15 ul sample were added in each reaction):

![](https://static.igem.org/mediawiki/2019/5/51/T--XJTLU-CHINA--table1.png)

![](https://static.igem.org/mediawiki/2019/b/b1/T--XJTLU-CHINA--table2.png)

**7. DNA Agarose Gel Electrophoresis**

When we apply MAXIscript Kit for the IVT of EAAT2-C/D Box-pcDNA3.1(+) and empty pcDNA3.1(+), we adjust the protocol to prevent the premature termination of transcription. To investigate if the prematuration is well-prevented, we run a DNA agarose gel electrophoresis and used 5 ul 1 kb DNA ladder as a length reference. We added 2 ul 6x DNA loading dye, 2 ul sample cDNA (1498.5 ng/ul), and add RNase-free water to 12 ul in each reaction.
We made a 1.0% agarose gel using 1 g agarose powder and 100 ml 1x TAE and 10 ul 10000x Gel-red. After the gel is cold, we loaded 10 ul sample in each well for electrophoresis.

**8. Exo-fect Exosome Transfection**

To transfect our mRNA into exosomes, we used SBI Exo-fect Transfection Kit, due to the limitation of reaction times, we used 30 ul Exo-fect solution to transfect 13.5 ug mRNA into approximately 9.37 x 107 exosomes. In order to thoroughly mix the reagents while the concentration of added exosomes are 10 times of the recommended, we extend the shaking time to 15 minutes and rise the shaking speed to 120 rpm. The transfected exosomes were then centrifuged at 21100 xg for 5 minutes, and 900 ul cold sterile PBS was added to resuspend the exosome pellet. The exosome solution was added to two N2a-culturing flasks with 2 ml DMEM exosome-free medium.

Our operation in this experiment follows the protocol for Exo-fect Transfection:
[Protocol link.](https://static.igem.org/mediawiki/2019/8/89/T--XJTLU-CHINA--Exo-fectTransfection.pdf)

**9. qRT-PCR**

24 hours after adding the exosome solution, we discarded the medium of two N2a cell-containing flasks, and washed them twice by using warm sterile PBS. The isolation of mRNA from those cells were conducted with application of Beyotime RNAeasy RNA Isolation Kit. The mRNA were then used as template in the following RT-PCR and qPCR procedures by applying the Cells-to-CT™ 1-Step Power SYBR™ Green Kit from Thermo Fisher. The results were then analyzed in Quantstudio 5 and relevant apps.

Our operation in this experiment follows the protocol for mRNA isolation:
[Protocol link.](https://static.igem.org/mediawiki/2019/8/86/T--XJTLU-CHINA--mRNAisolation.pdf)

Our operation in this experiment follows the protocol for qRT-PCR:
[Protocol link.](https://static.igem.org/mediawiki/2019/5/55/T--XJTLU-CHINA--qRT-PCR.pdf)

#### EAAT2-RNA Demonstration

This set of experiments is based on the completion of the exosome-delivery-relevant experiments.

**1. Cx43 Transfection**

In this part of experiment, the mass expression of Cx43 in both HEK293T and N2a cells was still essential for conducting the following procedures. We transfected Cx43-pcDNA3.1(+) both to HEK293T 3rd generations of cells and N2a 4th generations of cells in the same way (Engreen transfection) as that in the exosome-delivery experiments, which ensures the exosome to specifically deliver their cargo to neurons and glial cells expressing Cx43 on the membrane surface. Both of HEK293T and N2a cells were incubated in DMEM medium with 5% exosome-free FBS and 1% Penicillin-Streptomycin in two T25 flasks.

Our operation in this experiment follows the protocol for T25 plate engreen transfection:
[Protocol link.](https://static.igem.org/mediawiki/2019/7/73/T--XJTLU-CHINA--Transfection-Engreen.pdf)

**2. Ultracentrifugation: Exosome Isolation**

This procedure of isolating exosomes is the same as the ultracentrifugation method in the exosome-delivery experiments. 12 hours after the transfection of Cx43 in two HEK293T flasks, the medium was changed to exosome-free DMEM medium for cell culture. After 24 hours, the cell medium was removed for ultracentrifugation. The extracted exosome pellets were resuspended in 50 ul cold sterile PBS and well-stored in a -80 ℃ freezer.

Our operation in this experiment follows the protocol for ultracentrifugation: [Protocol link.](https://static.igem.org/mediawiki/2019/3/3c/T--XJTLU-CHINA--Exosome_Extraction-Ultracentrifugation.pdf)

**3. In Vitro Transcription**

We used MAXIscript Kit from Ambion for in vitro transcription of our isolated EAAT2-C/D Box-pcDNA3.1(+) and pcDNA3.1(+) (Endotoxin-free). The Ribo m7G Cap Analog (Promega) was also added in the reagent solution, which protects the naked mRNA and facilitate the translation of mRNA in N2a cells. After accomplishing the IVT, we added 1 ul TURBO DNase each sample to remove the template DNA in each sample, and incubated at 37 ℃ for 15 minutes.

Our operation in this experiment follows the protocol for in vitro transcription:[Protocol link.](https://static.igem.org/mediawiki/2019/b/bb/T--XJTLU-CHINA--InVitroTranscription.pdf)

**4. Polyadenylation**

In order to protect the naked mRNA transcribed and facilitate the translation of mRNA in eukaryotes, we used Poly[A] Enzyme from Thermo Fisher Advanced miRNA Assay Kit to add poly-A tails on the 3’ ends.

Our operation in this experiment follows the protocol for Polyadenylation:[Protocol link.](https://static.igem.org/mediawiki/2019/4/4d/T--XJTLU-CHINA--Polyadenylation.pdf)

**5. Transfection of EAAT2-C/D Box-pcDNA3.1(+) (mRNA)**

To ensure that the RNA transcribed from EAAT2-C/D Box-pcDNA3.1(+) can be well-expressed in N2a, we transfected the mRNA of part BBa\_k3030002 on pcDNA3.1(+) using engreen to P4 N2a cells. All of the Neuro-2a cells were incubated in 5% FBS-added DMEM medium with anti-anti in a 24-well plate until the cells were approximately 60% confluence.
To avoid the wrong results in WST-1 test caused by cytotoxicity induced by engreen, the empty pcDNA3.1(+) was used as the negative control plasmid, and transfected into another 12 wells, the distribution of sample in each well is displayed as below:

Our operation in this experiment follows the protocol for 24-well engreen transfection: [Protocol link.](https://static.igem.org/mediawiki/2019/7/73/T--XJTLU-CHINA--Transfection-Engreen.pdf)

**6. Cell Nurturing: Inflammation-inducing Condition**

This procedure of isolating exosome is the same as the inflammation-inducing step in exosome-delivery experiments. 12 hours after the transfection of both mRNA, the medium of N2a cells were changed to normal exosome-free DMEM medium.
To induce inflammation in the N2a cells, we changed the medium to phenol-free RPMI1640 medium with 5% exosome-free FBS, and additional glutamate were added to ensure that the concentration of glutamate is 80 uM.
The time interval of changing DMEM medium to glutamate-contained medium is controlled to three hours, and for both of the EAAT2-transfected and pcDNA-transfected N2a cells, there are four groups of cells to be manipulated, and each group has three wells. The distribution of sample in each well is displayed as below:

![](https://static.igem.org/mediawiki/2019/3/3e/T--XJTLU-CHINA--E10.png)

**7. WST-1**

The excitotoxicity induced by glutamate to N2a cells were detected by applying Beyotime WST-1 Cell Proliferation and Cytotoxicity Assay Kit. We added 20 ul WST-1 in each 200 ul-medium-containing well of the 24-well plate. After incubating the WST-1-added cells in 37 ℃ for 2 hours, we measured the absorbance at 450 nm, and 620 nm as reference wavelength in a 96-well plate.

Our operation in this experiment follows the protocol for WST-1 Test: [Protocol link.](https://static.igem.org/mediawiki/2019/b/bc/T--XJTLU-CHINA--WST-1.pdf)

**8. Glutamate Assay**

The measurement of glutamate’s concentration in cell medium is also the same as the glutamate assay procedure in exosome-delivery experiments. To detect the exact glutamate concentration in the cell medium that mimics the cerebrospinal fluid, we used Enzychrom Glutamate Assay Kit. The cell medium from each well was extracted, while 20 ul in each sample was used in the glutamate assay. The measurement of absorbance at 565 nm was conducted twice respectively: at the moment adding working reagent solution and after a 30-minute incubation at room temperature.

Our operation in this experiment follows the protocol for Glutamate Assay: [Protocol link.](https://static.igem.org/mediawiki/2019/3/3e/T--XJTLU-CHINA--GlutamateAssay.pdf)

#### Measurement-Part Improvement

**1. Transformation of 14O and 16A to DH5a**

After the plasmid containing BBa\_k598009, BBa\_k598010 from PKU 2011 part list, and our newly designed BBa\_k3030016, BBa\_k3030017 were well received, we conducted the transformation of these plasmids to DH5a competent cells respectively. 100 ul transformed cells were used to coat on LB plates and incubated at 37 ℃ overnight (12 hours approximately).

Our operation in this experiment follows the protocol for Transformation: [Protocol link.](https://static.igem.org/mediawiki/2019/3/3c/T--XJTLU-CHINA--Transformation.pdf)

**2. Miniprep**

To amplify the plasmid we received for the following experiments, we selected three colonies on LB-0.1% ampicillin selective plates respectively for the four plasmids. And incubated the 12 colonies in 10 ml LB-Amp medium for overnight.
Before the miniprep, we tested the OD600 for each colony using spectrometer, and the OD value were all above 2. Then we used Qiagen Miniprep DNA Isolation Kit to conduct the miniprep. The isolated DNA were then eluted using ddH2O, and stored in 200 ul centrifuge tubes in -20 ℃ freezers. For each plasmids, we used one tube (50 ul in approximately 150 ng/ul) of isolated DNA in the following transformation step.

Our operation in this experiment follows the protocol for Miniprep: [Protocol link.](https://static.igem.org/mediawiki/2019/f/f5/T--XJTLU-CHINA--QIAprepSpinMiniprep.pdf)

**3. Transformation of 14O and 16A to DE3**

The transformation of these four plasmids to DE3 was conducted after the miniprep of DE3 respectively. 100 ul transformed cells were extract and then incubated overnight (12 hours approximately) on LB-Amp plates.

Our operation in this experiment follows the protocol for Transformation: [Protocol link.](https://static.igem.org/mediawiki/2019/3/3c/T--XJTLU-CHINA--Transformation.pdf)

**4. Detection of GFP in induction system**

After the overnight incubation, the transformed DE3 colonies were selected and incubated in Sangon ready-for-use M9 minimal antibiotic-containing medium with MgSO4, CaCl2 and 20% glucose added. The incubation lasted for approximately 7 hours, and the OD600 reached 0.4.
The remaining steps were conducted as the protocol says for measurement and part improvement, and the distribution of the samples are displayed as below:

![](https://static.igem.org/mediawiki/2019/a/ae/T--XJTLU-CHINA--E11.png)

Our operation in this experiment follows the protocol for Measurement/Part Improvement: [Protocol link](https://static.igem.org/mediawiki/2019/b/b7/T--XJTLU-CHINA--RiboswitchTest.pdf).

### Notebook

[Please refer to our Notebook Page...](https://2019.igem.org/Team%3AXJTLU-CHINA/Notebook)

### Protocols

[Please refer to our Protocol Page...](https://2019.igem.org/Team%3AXJTLU-CHINA/Protocol)

### References

Beart, P. M. and Shea, R. D. O. (2007) ‘Transporters for L -glutamate : An update on their molecular pharmacology and pathological involvement’,  *British Journal of Pharmacology* , 150(1), pp. 5–17. doi: 10.1038/sj.bjp.0706949.

Das, S. K. et al. (2012) ‘Role of excitatory amino acid transporter-2 (EAAT2) and glutamate in neurodegeneration: opportunities for developing novel therapeutics’, *Journal of Cellular Physiology* , 226(10), pp. 2484–2493. doi: 10.1002/jcp.22609.

Emine, Ş., Solaro, I. and Gürsoy-özdemir, Y. (2018) ‘Cell Death Mechanisms in Stroke and Novel Molecular and Cellular Treatment Options’, *Current Neuropharmacology* , 16(9), pp. 1396–1415. doi: 10.2174/1570159X16666180302115544.

Falk, M. M. et al. (2016) ‘Molecular mechanisms regulating formation , trafficking and processing of annular gap junctions’, *BMC Cell Biology* , 17(1). doi: 10.1186/s12860-016-0087-7.

Foran E, Rosenblum L, Bogush A, Pasinelli P, T. D. (2014) ‘Sumoylation of the astroglial glutamate transporter EAAT2 governs its intracellular compartmentalization.’, *Glia* , 62(8), pp. 1241–1253. doi: 10.1002/glia.22677.Sumoylation.

Heiser, A. et al. (2002) ‘Autologous dendritic cells transfected with prostate-specific antigen RNA stimulate CTL responses against metastatic prostate tumors’, *The Journal of Clinical Investigation* , 109(3), pp. 311–312. doi: 10.1172/JCI200214364.Introduction.

Kanai, Y. and Hediger, M. A. (2003) ‘The glutamate and neutral amino acid transporter family : physiological and pharmacological implications’, *European Journal of Pharmacology* , 479(1–3), pp. 237–247. doi: 10.1016/j.ejphar.2003.08.073.

Levy, L. M., Warr, O. and Attwell, D. (1998) ‘Stoichiometry of the Glial Glutamate Transporter GLT-1 Expressed Inducibly in a Chinese Hamster Ovary Cell Line Selected for Low Endogenous Na ؉ -Dependent Glutamate Uptake’, *The Journal of Neuroscience* , 18(23), pp. 9620–9628.

Liu, B., Teschemacher, A. G. and Kasparov, S. (2017) ‘Astroglia as a cellular target for neuroprotection and treatment of neuro-psychiatric disorders’, *Glia* , 65(8), pp. 1205–1226. doi: 10.1002/glia.23136.

Morse, M. A. et al. (2002) ‘The Feasibility and Safety of Immunotherapy with Dendritic Cells Loaded with CEA mRNA Following Neoadjuvant Chemoradiotherapy and Resection of Pancreatic Cancer’, *International Journal of Gastrointestinal Cancer* , 32(1), pp. 1–6.

Sahin, U., Karikó, K. and Türeci, Ö. (2014) ‘mRNA-based therapeutics — developing a new class of drugs’, *Nature Publishing Group. Nature Publishing Group* , 13(10), pp. 759–780. doi: 10.1038/nrd4278.

Sitcheran, R. et al. (2005) ‘Positive and negative regulation of EAAT2 by NF- j B : a role for N-myc in TNF a -controlled repression’, *The Embo Journal* , 24(3), pp. 510–520. doi: 10.1038/sj.emboj.7600555.

Solan, J. L. and Lampe, P. D. (2018) ‘Spatio-temporal regulation of connexin43 phosphorylation and gap junction dynamics.’, *Biochemical et Biophysical Acta. Biomembranes* , 1860(1), pp. 83–90. doi: 10.1016/j.bbamem.2017.04.008.Spatio-temporal.

![](https://static.igem.org/mediawiki/2019/9/9a/T--XJTLU-CHINA--logogogogo.png)

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# Team:XJTLU-CHINA/Results

Xi'an Jiaotong-Liverpool University

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  PRACTICE](https://2019.igem.org/Team%3AXJTLU-CHINA/Human_Practices)
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* [EAAT2-Functional Characterization](#Title1)
* [C/D Box-L7Ae-Functional Characterization](#Title2)
* [Measurement-Part Improvement](#Title3)
* [Notebook](#Title4)
* [Protocols](#Title5)
* ![](https://static.igem.org/mediawiki/2019/5/5c/T--XJTLU-CHINA--Logo.png)

![](https://static.igem.org/mediawiki/2019/8/88/T--XJTLU-CHINA--results-new.png)

### EAAT2-Functional Characterization

At the beginning of our project, we characterized the function of EAAT2 and its expression, and simulated the inflammation-inducing environment for Neuro-2a cells using the high-glutamate condition exhibited in neurodegenerative patients’ cerebrospinal fluid.

In this part of test, we focused on two impacts our exosome medicine may bring to the patients:

* Reducing the excitatory amino acids in the cerebrospinal fluid.
* Alleviate the cytotoxicity brought by excessive glutamate or the excitotoxicity induced.

#### 1. Glutamate Assay

We tested two sets of cells: the EAAT2-transfected N2a cells and the empty plasmid pcDNA3.1-transfected cells (negative control). For each set of N2a cells, the time interval for changing the DMEM medium to inflammation-inducing medium was three hours. There were four groups (approximately 0 hour, 3 hours, 6 hours and 9 hours) in total which were the time to change the medium. The level of glutamate in the culture medium was then measured using the glutamate assay kit and the absorbance for each sample was read in a plate reader.

The distribution of each sample are displayed as below:

![](https://static.igem.org/mediawiki/2019/2/22/T--XJTLU-CHINA--R1.png)

![](https://static.igem.org/mediawiki/2019/2/27/T--XJTLU-CHINA--R2.png)

Figure 1. The expression of EAAT2 helps reduce the glutamate level in cell medium. The glutamate concentration in cell medium grows significantly faster in EAAT2-untransfected groups, which indicates that the expression of EAAT2 can help hold the concentration of extracellular glutamate.

![](https://static.igem.org/mediawiki/2019/7/76/T--XJTLU-CHINA--R3.jpg)

Figure 2. The glutamate concentration in pcDNA3.1(+)-transfected cell medium is significantly higher than those of EAAT2-transfected groups after six hours, Student’s t-test applied (\*: p<0.05, \*\*: p<0.01, \*\*\*: p<0.001).

The results of glutamate assay reveals that the expression of EAAT2 on cell membrane maintained the concentration of glutamate in a relatively stable level, and significantly reduced the concentration of glutamate in the cell medium. Higher the glutamate concentration is, the more significant EAAT2 removed the glutamate in cells. In addition, the expression of EAAT2 did not over-transport glutamate and maintained the glutamate concentration similar to the origin level.

#### 2. WST-1 Cytotoxicity Test

To detect the cytotoxicity induced by the excessively high glutamate concentration, we used the WST-1 assay as an alternative to the MTT assay for our test. The dehydrogenase in mitochondria can reduce the the tetrazolium salt WST-1 to soluble and chromatic formazan, and a high absorbance measured indicates a low cytotoxicity induced by glutamate. The absorbance of these samples were measured at 450 nm, and 690 nm was used as a reference wavelength.

The distribution of each sample are displayed as below:

![](https://static.igem.org/mediawiki/2019/9/93/T--XJTLU-CHINA--R4.png)

![](https://static.igem.org/mediawiki/2019/d/d8/T--XJTLU-CHINA--R5.png)

Figure 3. The cytotoxicity induced by high concentration of glutamate (in EAAT2-transfected cell medium) can be obviously alleviated as the A450-A690 is remarkably lower than that of pcDNA3.1(+)-transfected cells.

![](https://static.igem.org/mediawiki/2019/d/d9/T--XJTLU-CHINA--R6.png)

Figure 4. The comparison in A450- A690 between EAAT2-transfected and pcDNA3.1(+)-transfected Neuro-2a cells (after a two-hour incubation in 37 Celsius), Students' t-test was applied (\*: p<0.05, \*\*: p<0.01, \*\*\*: p<0.001).

The results of WST-1 indicated that the expression of EAAT2 on cell membrane could significantly alleviate the excitotoxicity induced by glutamate. The cytotoxicity may have limited influence on cell proliferation even if the cells are cultured in inflammation-inducing (80 uM glutamate) for longer than 9 hours.

### C/D Box-L7Ae-Functional Characterization

In this part of experiments, we investigated the function of L7Ae, which may binds the C/D Box on our designed mRNA, and anchor the therapeutic mRNA on the inner face of exosomes.

The wet lab focused on two impacts L7Ae may bring to the exosome medicine:

* The production of exosome in exosome donor (HEK293T cells)
* The RNA anchored in the exosome

#### 1. Nanoparticle Tracking Assay

After the engreen co-transfection of L7Ae with C/D Box-nluc/nluc/empty pcDNA3.1(+), we conducted NTA to detect if the transfection may influence the exosome production, and ensure that the concentration reached 2x105 /ul, which is the recommended exosome concentration in exosome transfection (Exo-fect) in the sample ultracentrifugated. We distributed the transfected HEK293T cells in a 12-well plate as below:

![](https://static.igem.org/mediawiki/2019/9/9d/T--XJTLU-CHINA--R7.png)

We sent 50 ul of exosome isolated from each set of cells, the results were sent back and listed below:

##### [*Result1*](https://static.igem.org/mediawiki/2019/8/82/T--XJTLU-CHINA--Result%281%292019.pdf)

##### [*Result2*](https://static.igem.org/mediawiki/2019/3/39/T--XJTLU-CHINA--Result%282%292019.pdf)

##### [*Result3*](https://static.igem.org/mediawiki/2019/9/9d/T--XJTLU-CHINA--Result%283%292019.pdf)

The original concentration of particles in three groups were all measured to be 1.3x1010 particles/ml, which is approximately 50x recommended concentration for exosomes to be transfected by Exo-fect.

For the particles with different diameters, the distribution of their concentration in each set of samples are listed as below:

![](https://static.igem.org/mediawiki/2019/2/2f/T--XJTLU-CHINA--R8.png)

Figure 5. The numbers of ~150-nm-diameter particles with different diameters measured by NTA in a 50 ul ultracentrifuged exosome solution were almost the same (of cells those transfected with (1) nluc+L7Ae (2) pcDNA3.1(+)+L7Ae (3) C/D Box-nluc+L7Ae), the solution was diluted with a factor of 300x.

These results shows that the co-transfection and expression of the plasmids listed would not affect the production of exosome production, the concentration of harvested exosomes with ~150nm diameter is about 1.08 x 109/ml.

#### 2. qRT-PCR

In the qRT-PCR experiment, we selected hsa-miR-23a-3p as the endogenous reference gene. We selected nluc as our target gene to test if the L7Ae may help to enlarge the volume of mRNA anchored on the inner surface of exosomes. We calculated the ΔCt values using the Ct of target gene (nluc) to be divided by Ct of endogenous reference gene.

The distribution of 96-well plate in qRT-PCR is displayed as below:

![](https://static.igem.org/mediawiki/2019/7/73/T--XJTLU-CHINA--R8.5.jpg)

After the qRT-PCR, we did not get any normal signal from the group of pcDNA3.1(+)+L7Ae, nluc targeted group. In terms of the rest two groups, we analyzed theΔCt as below:

![](https://static.igem.org/mediawiki/2019/9/9f/T--XJTLU-CHINA--R9.png)

Figure 6. The ΔCt values calculated in the groups with and without C/D Box transfected.The comparison in ΔCt values between C/D Box-nluc+L7Ae groups and no-C/D Box groups has eligible difference, which indicates that the transfection of L7Ae may have limited impact in increasing the mRNA enclosed by exosomes.The characterization of L7Ae and C/D Box will be repeated in future experiments.

### Measurement-Part Improvement

In this part of experiment, we repeated the experiments conducted by Peking\_R 2011 team. Characterized the part BBa\_k598009, BBa\_k598010, and our newly designed BBa\_k3030016 and BBa\_k3030017. In order to get a more significant results, we improved the protocols used by Peking\_R 2011 team, these improvements were well-documented in our protocols.

TWe focused on investigating the following points in our measurement-relevant experiments:

* The cytotoxicity induced by theophylline to DE3 cells.
* The absorbance of our cultured DE3 in different concentrations of theophylline in our experiments.
* The fluorescence intensity measured that indicates the sensitivity of riboswitch to theophylline.

#### 1. The growth curve of DE3 in different concentrations of theophylline.

We used four gradients of TPP (0 mM, 0.1 mM, 1 mM and 10 mM) to test if the addition of TPP may induce cytotoxicity to DE3, and influence the growth of them. Each single colony of the DE3 cells transfected with circularized pSB1A3 were cultured in 100 ul LB-0.1%Amp for 12.5 hours, the OD600 values were measured in each 15 minutes. We chose 12 colonies in this experiment, the cultured time was compared with the average calculated for the 12 colonies’ OD600. The growth curves of DE3 cells is displayed below:

![](https://static.igem.org/mediawiki/2019/c/c8/T--XJTLU-CHINA--RN1.png)

The growth curve indicates that the OD600 measured may be influenced by the concentration of TPP, and the DE3 incubated with 1 mM TPP grows best and the 10 mM TPP may significantly inhibit the growth of DE3 cells. But the difference in OD600 was not that remarkable when the incubating time is limited in 25000 seconds. Thus, we chose to culture the DE3 cells for 6 hours in the following experiments.

#### 2. Measuring the fluorescence intensity of DE3 cells transformed with four parts with standards and blank.

We repeated the Peking\_R 2011 experiment and adapted their arrangement of TPP gradients (0 mM without arabinose added, 0 mM, 0.1 mM, 0.3 mM, 1 mM, 3 mM, 5 mM, and 10 mM with 1 mM arabinose added). In addition, The absorbance measured was used in normalize the fluorescence intensity measured to minimize the interference caused by bacteria concentration, while the high number of colonies grew in each well may lead a wrong over-high fluorescence result. The absorbance is measured after the GFP fluorescence.

All the procedures were conducted under dark condition in a 96-well plate. The distribution of the colonies cultured is displayed as below:

![](https://static.igem.org/mediawiki/2019/a/ac/T--XJTLU-CHINA--RN2.jpg)

(The part number of XJTLU-Natural, XJTLU-Optimized, PKU-Natural, and PKU-Engineered are BBa\_k3030016, BBa\_k3030017, BBa\_598009, and BBa\_k598010 respectively)

The GFP fluorescence values were measured, the excitation and emission light was 485 nm and 535 nm respectively. To avoid the leakage of fluorescence interfering the reading of other wells, the time interval of measurement was extended to 20 ms. The exact GFP fluorescent intensity of each well is listed below:

![](https://static.igem.org/mediawiki/2019/f/f0/T--XJTLU-CHINA--RN3.png)

Figure 7. The dose responsive curves that manifesting GFP intensity measured at 535 nm divided by OD600 comparing to the TPP concentration (0 mM, 0.1 mM, 0.3 mM, 1 mM, 3 mM, 5 mM, and 10 mM), the concentration of arabinose in each well was controlled to be 1 mM. It can be shown that the GFP fluorescence intensity increases along with the concentration of TPP as the riboswitch was opened by the TPP.

![](https://static.igem.org/mediawiki/2019/f/f6/T--XJTLU-CHINA--RN4.png)

Figure 8. The GFP/OD600 measured comparing to TPP concentration in each well. Student’s t-test is applied with the figure (\*: p<0.05, \*\*:p<0.01, \*\*\*:p<0.001).

![](https://static.igem.org/mediawiki/2019/6/67/T--XJTLU-CHINA--RN5.png)

Figure 9. The working curves that indicates the sensitivity of the riboswitch designed to TPP. The activation ratio is fluorescence intensity under given TPP concentrations compared to that of without TPP. The TPP gradients were set as 0 mM, 0.1 mM, 0.3 mM, 1 mM, 3 mM, 5 mM, and 10 mM, the concentration of arabinose in each well was controlled to be 1 mM.

![](https://static.igem.org/mediawiki/2019/4/41/T--XJTLU-CHINA--RN6.png)

Figure 10. The activation ratio measured comparing to TPP concentration in each well. Student’s t-test is applied with the figure (\*: p<0.05, \*\*:p<0.01, \*\*\*:p<0.001).

The results shows that the riboswitch designed in part BBa\_k3030017 has a 10-time expression of GFP (normalized by OD600) comparing to that of BBa\_k598009, our improved part. In addition, the designed BBa\_k3030017 has a 11-fold activation ratio of the absence to presence of 10mM theophylline.

### Notebook

[Please refer to our Notebook Page...](https://2019.igem.org/Team%3AXJTLU-CHINA/Notebook)

### Protocols

[Please refer to our Protocol Page...](https://2019.igem.org/Team%3AXJTLU-CHINA/Protocol)

![](https://static.igem.org/mediawiki/2019/9/9a/T--XJTLU-CHINA--logogogogo.png)