iGEM 2020 — Team IISER_Berhampur

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# Team:IISER Berhampur/Notebook

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# Team:IISER Berhampur/Engineering

IISER-BPR IGEM

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# Introduction

Our proposed reporter system i.e. FRaPPe, is an engineered syn-bio device that can be used to accelerate drug development for various diseases. It is built by combining three different biological modules, a CID (Chemically Induced Dimerization) module, a FRET (Fluorescence Resonance Energy Transfer) module and a POI (Protein of Interest) module. Using the first two modules, we can deduce the efficiency of any peptide-based drug in inhibiting the interactions between the proteins present in the POI module in in-vitro conditions. Being an engineered device, it's building, testing and improving process is based on several rounds of the engineering design cycle, but due to the constraints placed by the COVID-19 pandemic, we could only proceed to a small extent in the actual implementation cycle. Most of our design cycle has been based on suggestions from experts and our own molecular modelling and simulations, which is described below. However, we will be describing all that we have achieved in these terms and hope to achieve in the future in the following sections.

Research
Imagine
Design
Build
Test
Learn
Improve

[DRUG\_DEVELOPMENT](#block1) [INSIGHTS\_INTO\_INTERACTIONS](#block2) [REVIEW\_OF\_INHIBITORS](#block3)

# RESEARCH

![](https://static.igem.org/mediawiki/2020/7/77/T--IISER_Berhampur--_eng101.png)

# 1. DRUG DEVELOPMENT

To understand the predicament faced by humanity in developing effective therapeutics against deadly diseases, one need not look further than the COVID-19 pandemic waging around us right now. The existing notion is that it takes at least 10 years for a drug to be discovered and commercialised. The bitter truth : the actual journey might even take three times this time period.

***We first assessed the current drug development scenario.***

A new discovery takes several years to be refined and matured into a viable product. For viruses, in particular, the problems are many-fold. The arsenal is much more limited when compared to other infectious agents such as bacteria. Moreover, since viruses are technically not alive, they hijack the cells of our own body to feed their replication machinery. Any drug that targets a part of its life cycle, could end up harming the host in the process. And then comes the issue of diversity among viruses. Investing a huge deal of money and resources to study every unique virus stretches out the time needed immensely.

![](https://static.igem.org/mediawiki/2020/a/a1/T--IISER_Berhampur--_eng2.png)

**Figure 1:** Drug Discovery and Development : A Timeline. Information sourced from U.S. Food and Drug Administration. **https://www.fda.gov**

Specifically for Dengue, our disease of interest, the existence of different serotypes makes it very difficult for researchers to come up with a pan-serotype drug. Currently there are no clinically approved drugs against dengue. Until now most drugs that went into trial were repurposed from treatments of other diseases. For example Balapiravir which was initially developed as a drug against hepatitis C virus was entered into clinical trials but failed to show any significant effect when compared to placebos. (Shi et al., 2014)

# 2. INSIGHTS INTO INTERACTIONS

***We want to solve the Dengue scourge. The first order of business will therefore be to find what is so special about this virus that can give us an edge over conventional supportive therapies.***

As it turns out, Dengue is one of the masters of an intricate phenomenon named Protein-protein interactions (PPI). In this, two proteins in close proximity come in contact with each other in a certain orientation, forming a complex (either transient or permanent) in order to perform a specific function. In a cell, interactions between the various proteins occur at many different levels and bring about numerous cellular processes and pathways. As such PPIs form a very integral process in living cells.

Especially during a viral infection, the virus can only survive and multiply in the host system by making use of numerous protein-protein interactions between its proteins and the proteins of the host cell. These host-virus PPI’s serve to perform various functions from facilitating virus entry into the cell, to disabling host immune response pathways and spreading through tissues. When talking specifically about Flaviviruses like DENV, which are RNA viruses, PPIs are employed by the virus on every step of its pathogenesis. For example, right from the point when the viruses are being secreted from the infected cells, the mature virus is composed of multiple copies of core and capsid proteins, two envelope proteins and a virus-specific set of host proteins that enables it to be properly secreted so that it can then enter and thereby infect other cells. During entry into the cells, the primary protein interactions at the cell surface either serve to concentrate virus particles or trigger the productive virus uptake program. The host factors that flaviviruses generally tend to interact with are - heparin sulfates, Dendritic Cell-Specific Intracellular Adhesion Molecule (ICAM)-3-Grabbing Non-Integrin 1 (DC-SIGN, CD209 antigen), and DC-SIGNR, which interact with N-linked glycans of the viral E glycoprotein. Also, after the cleavage of the polyproteins and formation of functional protein subunits, these proteins induce the rearrangement of the ER membrane to form membranous compartments to facilitate viral replication and assembly. Here too, host-viral PPIs are made use of to form a replication complex, stabilize it and then disable the immune response of the host. Specific examples of these interactions include - interactions between DENV NS5 and GBF1 which induces ER membrane remodelling and replication complex formation early in the DENV infection state and also those between NS4B and 12 mitochondrial proteins that induce changes of the mitochondrial architecture which is necessary to prevent viral RNA recognition by the innate immune system. Sometimes the structural proteins to help in affecting immune responses other than just facilitating entry. For example, the capsid proteins of DENV and WNV have been found to interact with host peroxisome biogenesis factor- Pex19. This leads to loss of peroxisomes in the cell which diminishes early antiviral signalling and formation of interferons. (Gerold et al., 2017)

There are many other such host-viral PPIs that are absolutely essential for entry and replication of viral particles in the cell. Thus, inhibition of host-virus PPI is a therapeutic technique that holds potential in order to develop an effective antiviral drug against Dengue and all other viral diseases where this is a common theme.

# 3. REVIEW OF INHIBITORS

***We also compared the different options available among the classes of molecules widely used as drugs before zeroing in on our optimal molecule.***

![](https://static.igem.org/mediawiki/2020/e/ec/T--IISER_Berhampur--_eng3.png)

**Figure 2:** Comparison of the existing inhibitor drug molecule classes. Bruzzoni-Giovanelli et al. 2018 ; Lau et al., 2018 ; Lee et al., 2019 ; Sorolla et al., 2019

We can see that peptides are the preferred choice for our purpose. However, they have some disadvantages too. Peptides are generally unstable and can be degraded easily, but several studies done in the last few decades paved the way to get rid of these problems through methods like termini protection and chemical modifications. Also, there are recent advances in techniques to overcome several other intrinsic disadvantages of peptide drugs such as their solubility, low membrane permeability, fast elimination and bioavailability. That is the reason why, over the past two decades, nearly 60 peptide drugs have been approved worldwide. iPEPs as biotherapeutics are therefore receiving increasing attention.

![](https://static.igem.org/mediawiki/2020/a/a1/T--IISER_Berhampur--_exp100.png)

**Figure 3:** Cumulative number of peptides approved in major pharmaceutical markets and the number of peptides entering clinical development. Entry into clinical development is defined as the year of the first Phase 1 or pilot human study (Lau & Dunn, 2018).

 Next

[PPI\_REPERTOIRE\_OF\_DENV](#2block1) [THE\_TARGETS](#2block2) [ENVISAGING\_FRaPPe](#2block3)

# IMAGINE

![](https://static.igem.org/mediawiki/2020/0/07/T--IISER_Berhampur--_eng102.png)

# 1. PPI REPERTOIRE OF DENV

One of the important mechanisms of dengue viral pathogenesis manifestation is through myriad protein-protein interactions of viral Non-structural (NS) and host proteins. Antiviral approaches explored thus far have targeted both structural and nonstructural proteins of DENV. The structural proteins are the molecules that the virus particle comes equipped with, such as the proteins comprising their capsids or coats. The non-structural proteins are encoded by the viral genome and expressed in the infected host cells. These are mostly important players in replication and translation processes. We propose to design biomolecules which targets NS proteins specifically and disrupts all their pathogenesis pathways. (Visit our modelling section to know why we chose NS proteins using mutational analysis.)

The Dengue virus boasts a repertoire of seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5) coded for by its 10.7 kilobase capped positive sense, single-stranded RNA genome. These are all formed as a single polyprotein, and then chopped up into functional parts by the host and viral enzymes. They are the workhorses for viral replication and can also temper host immune responses in clever ways. So which one to choose? For this, first, we did an extensive literature survey to find out various PPI associated with each of the 7 DENV NSPs and collected structural and functional data about these interactions. From these we formed representative interactomes for each DENV NSP and finally, based on several criteria such as the amount of structural data available for each PPI and importance of the function each PPI plays in DENV Pathogenesis, we finally narrowed it down to the interaction between DENV NS5 and hSTAT2. (Read more about this in our [**Modelling section**](https://2020.igem.org/Team%3AIISER_Berhampur/Model).)

# 2. THE TARGETS

The relevance of this PPI was based on its involvement in the interferon (IFN) signalling pathway, a primary component of the innate antiviral response in mammals. To understand this, we did another round of research. The type 1 interferon (IFN) pathway acts to delay virus replication and to stimulate the activation of antiviral effector cells. The IFN receptor functions through the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway in which JAKs phosphorylate STATs, which then translocate to the nucleus to induce the expression of IFN-stimulated genes (ISGs). Type I IFN signalling activates phosphorylated STAT1 and STAT2 to heterodimerize and associate with interferon regulatory factor-9 (IRF9) to form IFN-stimulated gene factor-3 (ISGF3), which specifically binds IFN-stimulated response elements (ISREs) within antiviral gene promoters. Over the past decade, flavivirus NS5 proteins have surfaced as potent antagonists of IFN signalling.

DENV proteins NS2A, NS4A, and NS4B are able to antagonize IFN signalling. NS4B is highly potent and prevents STAT1 phosphorylation. DENV NS5, on the other hand, binds to STAT2 and inhibits IFN dependent signalling, by targeting STAT2 for proteasome-mediated degradation only when it is expressed as a proteolytically processed precursor. More importantly, The loss of STAT2 expression is conserved across the DENV serotypes and it occurs independently of the structural proteins.

![](https://static.igem.org/mediawiki/2020/c/c2/T--IISER_Berhampur--_eng6.png)

**Figure 4:** Diagrammatic Representation of the JAK-STAT pathway involved in the IFN response and inhibition of components by non-structural proteins of DENV. The inset shows the ubiquitin-mediated proteasomal degradation of STAT2 initiated by NS-5.

Further evidence to consolidate the role of STAT2 as an important player in IFN responses come from studies by Perry et al., 2011 which showed that STAT2 mediates STAT1-independent protection against DENV infection in mice. The combined loss of STAT1 and STAT2 results in high viral burden in tissues. It has been demonstrated by the same research group that mice lacking STAT1, a key mediator of both type I and II IFN responses, are not susceptible to DENV-mediated disease. So, what is the mechanism responsible for protection against DENV disease in the absence of STAT1? STAT2 seems to be the protein that mediates type I IFN signalling during DENV infection in the absence of STAT1. The resulting antiviral response includes amplification of type I IFN and the expression of interferon-stimulated genes (ISG) by associating with their promoter elements and leading to the antiviral response.

# 3. ENVISAGING FRaPPe

Hence, our aim now, is to study the Protein-protein interaction between the NS-5 viral protein and hSTAT2 host protein. How to do this?

We thought of using inhibitory peptides. And, gradually the idea formed of developing a composite reporter that can kill two birds with one stone: it can help the biologist study the fundamental molecular interaction of the proteins and can aid the chemist design an appropriate drug and screen its efficiency as well.

 Next

[CHEMICALLY\_INDUCED\_DIMERIZATION](#3block1) [FRET](#3block2) [COMBINED\_CID-FRET](#3block3)

# DESIGN

![](https://static.igem.org/mediawiki/2020/9/97/T--IISER_Berhampur--_exp103.png)

# 1. Chemically Induced Dimerization

In order to study our target interaction, we would need a viral infected cell. This is where Synthetic Biology came to our rescue. Instead of actual viral infection (which would require specialized laboratory set up and additional safety protocols), we decided to assemble the viral protein and the host protein in plasmid constructs. FRaPPe this helps us achieve the replication state in mammalian cells without needing patient samples.

**Module 1** : CID

**Purpose** : Bring the two proteins in proximity and keep control on their interaction.

**Challenge** : The proximity and the strength of the interaction between the proteins may be difficult to control or modulate.

**Solution** : We decided to use our first weapon, which is a chemical biology technique called, Chemically Induced Dimerization, wherein two particular proteins which can be induced to dimerize with one another, are fused with our selected proteins and expressed. In this way, our chosen proteins can be made to interact by bringing them in close proximity. Thus, we can replicate the viral infection with respect to host-viral protein interaction in a relatively safe setting.

In our system we aim to use FKBP12 and FRB protein domains and Rapamycin, that will induce their dimerization (Chemical Inducer of Dimerization). With the CID Reporter, we can ensure that our chosen PPI takes place in the cell and we can localize and observe it more closely.

![](https://static.igem.org/mediawiki/2020/e/ee/T--IISER_Berhampur--_eng8.png)

**Figure 5:** The binding affinities of the different components in the FKBP-Rapamycin-FRB system. Binding affinities are greatly increased in the presence of Rapamycin and a ternary complex is formed via two possible routes. Kd values taken from Banaszynski et al., 2005

# 2. Fluorescence or Förster Resonance Energy Transfer

**Module 2**: FRET

**Purpose** : Detect PPI and offer a quantifiable measure of strength of interaction in presence or absence of Peptide Inhibitor.

**Challenge** : To quantify the extent of the interactions, we need a second missile, a readout of the interactions.

**Solution** : We plan on using a FRET Reporter as an assay system to understand the interaction. From the literature survey, it was clear that PPI followed by Immuno pull down and western blotting will be time consuming. And if we wanted an immediate readout, fluorescent tagged proteins could do the trick. FRET (Forster’s or Fluorescence Resonance Energy Transfer) is a phenomenon that essentially functions as a spectroscopic ruler, by employing two fluorophores (donor and acceptor) wherein the excitation energy of the donor is transmitted to the acceptor in its vicinity, resulting in the distinctive fluorescence emission spectra of the acceptor.

Such biosensor systems are highly sensitive to the distance of separation between the interacting molecules (within 1 to 10nm range). Due to this property, FRET is used to quantify many biological processes that are dependent on molecular proximity such as Protein-Protein Interactions. FRET is more advantageous than other Reporter systems because it allows observation of live cells in a non-destructive and minimally invasive way. Efficiency can be used as a measure of quantification.

The FRET Efficiency is basically the percentage of energy transfer from the donor to acceptor fluorophores at a given state and depends on the distance between donor and acceptor dipoles, while the FRET Dynamic Range refers to the range at which the reporter operates and is essential for detection of highly sensitive cellular events.

# 3. COMBINED CID-FRET

In our project, we are going to use a FRET-CID coupled system where, after the CID reporter ensures the PPI, the FRET reporter will measure the extent of interaction and also act as a measure for the peptide inhibitor efficiency by showing a change in the acceptor fluorophore’s emission spectra. The CFP will be our donor fluorophore and YFP, our acceptor fluorophore. The measurable parameters generated from the FRET system, namely the efficiency and dynamic range will be our tools in gauging the interaction and its subsequent inhibition.

![](https://static.igem.org/mediawiki/2020/e/ea/T--IISER_Berhampur--_eng9.png)

![](https://static.igem.org/mediawiki/2020/7/7f/T--IISER_Berhampur--_eng10.png)

![](https://static.igem.org/mediawiki/2020/6/64/T--IISER_Berhampur--_eng12.png)

**Figure 6:** Schematic diagram of the building blocks of FRaPPe. Modules used include CID, FRET and POI.

 Next

[FRaPPe\_CONSTRUCTS](#4block1) [PLANNED\_DESIGNS](#4block2) [BUILDING\_OUR\_BIOBRICKS](#4block3)

# BUILD

![](https://static.igem.org/mediawiki/2020/7/73/T--IISER_Berhampur--_exp104.png)

# 1. FRaPPe CONSTRUCTS

To assemble the modules into our plasmid constructs we designed several possible combinations. The molecular docking experiments were the starting point. Using the visualization of the structure based on the experimental data, we chalked out a rough design for our plasmids. Since the experimental data was limited, and did not resemble the entire length of the proteins, we used the reconstructed structure using homology modelling to obtain a clearer picture. This gave us insights about selecting appropriate N or C terminals to position our FRET fluorophores. (To read more about this, visit our [**Modelling section**](https://2020.igem.org/Team%3AIISER_Berhampur/Model)).

# 2. PLANNED DESIGNS

Using these inputs we narrowed down our constructs from an initial 36 to 10. These are shown in the constructs table below along with their respective codes.

| CODE | CONTROL CONSTRUCTS | CODE | TEST CONSTRUCTS |
| --- | --- | --- | --- |
| FRaPPe C1 | FKBP-CFP | FRaPPe T1 | FKBP C-hSTAT2(FL)-N CFP |
| FRaPPe C2 | FRB-YFP | FRaPPe T2 | FRB C-NS-5(FL)-N YFP |
| FRaPPe C3 | C-hSTAT2(FL)-N CFP | FRaPPe T3 | CFP FKBP N-hSTAT2(FL)-C |
| FRaPPe C4 | C-NS-5(FL)-N YFP | FRaPPe T4 | YFP FRB N-NS-5(FL)-C |
| FRaPPe C5 | FKBP-N-hSTAT2(FL)-C |  |  |
| FRaPPe C6 | FRB-N-NS-5(FL)-C |  |  |

# 3. BUILDING OUR BIOBRICKS

* For our first set of constructs, we will insert our genes of interest in a suitable CID plasmid backbone, so as to create fusion constructs, FRaPPE C5 and FRaPPe C6 (See construct table).

* The interactions between any DENV NS and host protein or between two DENV NS proteins can be studied by ligating their coding sequences with the CID Reporter.

* The plasmid will be cloned in E. coli and the resulting reporter system will be expressed in HEK-293 cell lines. We have chosen HEK293 cells due to their ease of transfection and maintenance in culture. They have a rapid doubling time (36 hours) and can be reliably used for both stable and transient transfection (we will be doing the latter). Thus, compared to more cancerous cell lines like HeLa, HEK cells are the more feasible choice for studying our reporter in a mammalian system.

* In addition to the previous CID reporter, the DENV NS protein and its host target will be ligated with CFP and YFP respectively. Test construct series FRaPPE T1-T4 will be generated to account for different orientations. We also propose the control constructs FRaPPE C1-C4 to take into account effects of the CID alone and the interaction between our proteins of interest in the absence of CID. (See Construct Table)

* These plasmids will also be cloned in E. coli and the whole system, in turn, will be transfected into HEK 293 cell line, where it can be used as the control for the assay platform for screening of small molecules/activators/peptide inhibitors.

 Next

[PLASMID\_FINALIZATION](#5block1) [RESTRICTION\_DIGESTS](#5block2) [CLONING\_AND\_TRANSFECTION](#5block3) [PEPTIDE\_INHIBITOR\_EFFICIENCY](#5block4)

# TEST

![](https://static.igem.org/mediawiki/2020/f/f4/T--IISER_Berhampur--_exp105.png)

# 1. PLASMID FINALIZATION

Since we did not have access to lab facilities during the lockdown, we were not able to carry out our wet lab completely. We have started our preliminary experiments recently. (Refer to [**Experiments Section**](https://2020.igem.org/Team%3AIISER_Berhampur/Experiments)). Nevertheless, we did plan how we would be conducting the experiments if the opportunity came up.

* We ordered CID-FRET backbone plasmids from Addgene [Plasmid #20160, CFP-FKBP (CF) and Plasmid #20148, YFP-FRB (YR)], which contain the coding sequences of CFP and YFP fluorophores that we have decided to use as our FRET pair, in addition to coding sequences for FKBP and FRB (FKBP-Rapamycin Binding Domain) under the control of a CMV promoter and enhancer. It used Neomycin/Kanamycin resistance as a marker. Coding sequences of POIs were ordered from Twist Bioscience.

* Based on the Restriction Enzyme sites available in the plasmids at the appropriate locations for inserting our POI modules, we chose those which were not present within the coding sequences of NS-5 and hSTAT2 to avoid fragmentation during cloning. We modified the gene sequences for our Proteins of Interest as follows:

* For NS-5 (GenBank : KR919821.1, DENV TSV08 Serotype 1) . To be cloned into Addgene plasmid # 20148 using HindIII at 5’ and SalI at 3’

* For hSTAT2 (GenBank : U18671.1) . To be cloned into Addgene plasmid # 20160 using XhoI at 5’ and EcoRI at 3’

* Plasmid maps were prepared accordingly. (See [**Parts Section**](https://2020.igem.org/Team%3AIISER_Berhampur/Parts/Part_Collection))

* Since the coding sequences of the proteins are quite large, we were advised by Twist Bioscience to use Splicing by Overlap Extension to get them synthesized, hence we modified the strategy accordingly.

# 2. RESTRICTION DIGESTS

* Once all our reagents and components arrive, we will first be performing the above mentioned restriction digests for the corresponding plasmids as per our constructs table. This will be followed by ligation to create our test constructs. For our control constructs, similar procedures will be used. In C3 and C4, the modules for CID will be excised out by appropriate restriction digests and the gene blocks inserted instead. For C5 and C6, the fluorophore sequences will be excised and replaced by gene blocks.

* Given that the YR plasmid has an MCS between the FRB and YFP sequences, construct T2 will be made, and for the CF plasmid, insertion between FKBP and CFP may not be reliable due to absence of an MCS, hence construct T3 will be made.

# 3. CLONING AND TRANSFECTION

* Transformation of the plasmid into competent DH5α E. coli cells will follow and positive recombinants for FRaPPe constructs will be confirmed by colony PCR or restriction digestion and sequencing.

* Confirmed plasmids will be amplified by Maxiprep to prepare DNA for transfection into HEK293 cells. For test cell lines, constructs T2 and T3 will be co-transfected, and for control cell lines, constructs C1, C2 ; C3, C4 ; C5, C6 will be pairwise co-transfected in different cultures. Presence of reporter will be screened by qPCR.

* Cells will then be used for a dose dependent Rapamycin assay and corresponding FRET measurement to choose an optimum Rapa concentration.

* After procuring the chemically synthesized peptide inhibitor, the FRaPPe T2 and T3 co-transfected cells will be treated with the inhibitor and the efficiency of each peptide inhibitor in inhibiting the PPI will be quantified. The various approaches for this quantification are discussed in the section below.

![](https://static.igem.org/mediawiki/2020/a/ad/T--IISER_Berhampur--eng1111.png)

**Figure 7:** Overview of the Reporter System showing the overall test procedure for experimentation and characterization of its working.

# 4. PEPTIDE INHIBITOR EFFICIENCY

For the quantification of peptide inhibitor efficiency, the following 2 approaches have been suggested-

* **Approach 1** - This approach focuses on the quantification of the inhibition based on the FRET readout with respect to different concentration of peptide inhibitors

* **Approach 2** - This methodology focuses on controlling the level of Rapamycin for ranking of the peptide inhibitors.

## Approach 1

* Step 1 : Initially FRaPPe is kept at a fixed concentration of Rapamycin ( Assumed to be x) .
* Step 2 : Now we titrate the cell culture containing our reporter system with the peptide inhibitor and note the FRET emission after each addition. From here we obtain a plot of FRET emission versus the log(concentration) of the peptide inhibitor.
* We can then obtain the concentration of the peptide inhibitor for which the FRET emission gets reduced by 50% of the initial value (without iPEP) which is the IC50 value. IC50 values for each inhibitor can then be compared and ranked.

![](https://static.igem.org/mediawiki/2020/0/02/T--IISER_Berhampur--_eng99.png)

**Figure 8:** Representative diagram for obtaining IC50 values

## Approach 2

Since the reporter system consists of a CID system in which the interactions between FKBP-FRB are mostly regulated by the addition of Rapamycin, it can act as a good indicator to find out the efficiency of the peptide inhibitors and rank them accordingly.

* Step 1 : The basic principle behind this method is that with increasing concentration of Rapamycin, we can increase the amount of FKBP-FRB dimerization that takes place and hence increase the FRET efficiency values readout. Initially, with some amount of rapamycin (assumed to be x) we will keep the FRET efficiency at a certain fixed value.
* Step 2 : A certain concentration ( assume it to be z) of peptide inhibitor is added to the system. Now the concentration of the Ramamycin is increased until the FRET efficiency value reaches back to our fixed value .

Since with the addition of peptide inhibitors the interactions between the proteins will be inhibited, to reach the initial FRET efficiency value, more amount of Rapamycin will be required. Let it be x+y. Then the value of y gives us a proxy of the efficiency of the peptide inhibitors. A higher value of y will indicate that the peptide is a better inhibitor of the PPI.

 Next

[EXPRESSION](#6block1) [TRANSFECTION](#6block2)

# LEARN

![](https://static.igem.org/mediawiki/2020/b/ba/T--IISER_Berhampur--_exp106.png)

We have tried to analyse the points where we can encounter possible challenges during our reporter creation.

# 1. EXPRESSION

Potential challenge: Since there is a co-transfection, the expression level of the two proteins may show differences.

Solution: The expression and localisation pattern of the proteins in the engineered cells will be first understood using the control constructs.

# 2. TRANSFECTION

Potential challenge: Transfection, our method to introduce the plasmids into HEK cells, may generate heterogeneity as not all cells will take up the plasmids uniformly.

Solution: We plan to use electroporation after preliminary optimization with the transfection.

 Next

[OPTIMISING\_TRANSFECTION](#7block1) [ROLE\_OF\_CID](#7block2) [REFERENCES](#7block3)

# IMPROVE

![](https://static.igem.org/mediawiki/2020/3/3f/T--IISER_Berhampur--_exp107.png)

Based on the anticipated challenges that we may face, some possible improvements that we can introduce have also been brainstormed. Many of these inputs were provided to us in different interviews with researchers working in CID, FRET and related fields

# 1. OPTIMISING TRANSFECTION

Potential challenge: Transient transfection may not provide reliable results while working with a CID system. Control over expression.

Solution: Lentiviral based transduction approach for generating stable cell lines and also for introduction of a single construct with both the POIs separated by linkers.

Strategy: See construct info of FRaPPe T1 and T2, the gene hSTAT2 and NS5 have been synthesized with additional REs AgeI and MluI to facilitate easy excision and introduction to lentiviral vector (addgene 17446, pLenti CMV GFP Hygro).

# 2. ROLE OF CID

Potential challenge: Use of CID for the inhibitor screening system.

The FKBP-FRB based CID system is considered to be a very strong interaction system. (See Figure 4 under design tab). The Kd values reflect an irreversible interaction, and hence it is possible that they can affect the interaction between the POIs (in terms of peptide efficiency determination).

To elaborate, based on FRaPPe constructs T2 and T3, the FRET efficiency readout will actually be a function of two interactions:

(i) FKBP-FRB interaction

(ii) NS5-STAT2 interaction

But suppose, the interaction between FKBP-FRB dominates strongly over the NS5-STAT2, then the FRET readouts may not decrease even if the peptide inhibitors are working very efficiently in inhibiting interaction between NS5 and STAT2.

Solution: Use of control constructs to identify the relevant interaction and corresponding inhibition.

Strategy:

* The control constructs FRaPPe C1, C2, C3, C4 will help in solving the above problem.
* Additionally, rapamycin concentration can be titrated to modulate the FKBP-FRB interaction.
* In FRaPPe T1 and T2 CFP and YFP are attached directly to NS5 and hSTAT2 and not FKBP and FRB, thus may not be influenced by the strength of FKBP-FRB interaction alone.
* An alternative reversible CID system which can bring the target proteins into proximity but will not affect their interactions significantly. While such systems are not very widely available, research has revealed some systems that can suit this purpose, such as that using a synthetic ligand of FKBP (SLF’). SLF’ binds to FKBP and is also capable of binding E.coli dihydrofolate reductase (eDHFR) via its TMP (Trimethoprim) moiety. The rapid SLF’-TMP induced heterodimerization of FKBP and eDHFR can be easily reversed by using TMP itself as a competitive inhibitor. (Liu et al., 2014)

However, our Molecular Dynamics Simulation (visit our [**Modelling section**](https://2020.igem.org/Team%3AIISER_Berhampur/Model)) shows that the interaction of NS5 and hSTAT2 is relatively weak, thus strengthening the need for a regulated mechanism of PPI. The use of CID should circumvent this problem and hence we are making an informed choice of using the CID system for creating FRaPPe.

# REFERENCES

1. Banaszynski et al. (2005) Journal of the American Chemical Society, 127(13), 4715-4721.

2. Bruzzoni-Giovanelli et al. Drug Discovery Today 23.2 (2018): 272-285.

3. Gerold, G. et al. (2017) Molecular & cellular proteomics : MCP, 16(4 suppl 1), S75–S91.
[**https://doi.org/10.1074/mcp.R116.065649**](https://doi.org/10.1074/mcp.R116.065649)

4. Gu F, Shi Y (2014). Clin. Invest. (Lond.) (2014) 4(8), 683–685.

5.Lau et al. Bioorganic & medicinal chemistry 26.10 (2018): 2700-2707.

6. Lee et al. International journal of molecular sciences 20.10 (2019): 2383.

7. Liu et al. (2014) Angewandte Chemie International Edition, 53(38), 10049-10055.

8. Sorolla et al. Oncogene (2019): 1-18.

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# Team:IISER Berhampur/Experiments

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[Preliminary\_Standardization](#block1) [Transformation](#block2) [RE\_Digestion](#block3) [Cell\_Culture](#block4) [Notebook](#block5)

# Work Outline

Due to the COVID-19 pandemic, we had to re-strategise our project. Owing to the lockdown, the team could not access the lab till the beginning of October. For the same reason, reagents could not reach us on time either.

However, we decided to utilize this time in planning and revisiting our experiments thoroughly.

* literature reviews, ([**Engineering Success**](https://2020.igem.org/Team%3AIISER_Berhampur/Engineering)  section)
* discussions with experts in the field ([**Human Practices section**](https://2020.igem.org/Team%3AIISER_Berhampur/Human_Practices))
* computational modelling ( [**Modelling section**](https://2020.igem.org/Team%3AIISER_Berhampur/Model)).

# Preliminary Standardization of Plasmid

And these made us realise that instead of waiting for the reagents to arrive and then start optimizing protocols and techniques, we should start working on trial experiments to optimize our cloning, transfection and FRET approaches, as soon as we could access the lab.

We were able to do this with the help of a FRET reporter (Bulusu et al., 2017), that was available in the Department of Biological Sciences (DBS), IISER Berhampur.

All the protocols below have now been optimized and we plan to use this for our constructs for the second phase of the project.

## Details of our Optimization Plasmid

pCKI – PYRATES

The pCKI vector is a modified pCAGGS (CMV-enhancer, beta-actin promoter, beta-globin polyA) ubiquitous expression vector. This backbone contains the coding sequence for variants of CFP (mTurquoise) and YFP (cp173Venus) and Ampicillin resistance as a selectable marker. The plasmid was kindly gifted to us by Dr. Vinay Bulusu (Assistant Professor, DBS), IISER Berhampur.

![](https://static.igem.org/mediawiki/2020/f/fd/T--IISER_Berhampur--_pck1.png)

**Figure 1** Plasmid Map showing the components of the pCKI plasmid with the coding sequences of CFP and YFP variants.

## In Silico Restriction Digests

### Overview

We performed in silico digests with the circular pCKI sequence using NEBcutter V2.0 ([**http://nc2.neb.com/NEBcutter2/**](http://nc2.neb.com/NEBcutter2/) ) to obtain virtual gel images and find the non-overlapping open reading frames in the obtained plasmid. We assumed usage of a 1.0% agarose gel, to determine the mobility of known size fragments based on existing experiments in the NEB database. Three possible sets of high fidelity restriction enzymes were used (See Results below). The mobility of the fragments resulting from custom digests are predicted by polynomial interpolation from the standard cubic spline curve for known digests and the expected gel image is generated which can be matched with experimental digests.

Since the pCKI plasmid contains coding sequences of the CFP and YFP variants, we decided to excise out these fragments for additional FRaPPe constructs. In silico digests to release these fluorescent reporters was performed. We aim to use these variants as they are more stable forms than conventional CFP and YFP. mTurquoise is one of the brightest and most bleaching-resistant FRET donor fluorophores with a high quantum yield while cp173 Venus shows a high FRET efficiency. This pair also prevents the formation of bright aggregates of sensors, or speckles which can impede accurate measurement. (Klareenbeek et al., 2015)

This will enable us to use the pCKI plasmid both for optimization of our cloning and transfection protocols as well as for reporter construction. By using these variants in place of the existing sequences in Addgene plasmids (#20148 and #20160) we will modify them to carry an enhanced FRET sensor.

### Results

### 1. Restriction Enzyme Digestion Map for pCKI, all commercially available NEB enzymes.

![](https://static.igem.org/mediawiki/2020/1/1f/T--IISER_Berhampur--_exp2.png)

**Figure 2** pCKI RE digest Map

### 2. Custom Restriction Digests were done using the following sets of enzymes.

* HindIII and EcoRI Double Digest
* HindIII and BamHI Double Digest
* BamHI Single Digest

![](https://static.igem.org/mediawiki/2020/9/95/T--IISER_Berhampur--_exp3.png)

**Figure 3a** Circular Digests and ORFs generated

![](https://static.igem.org/mediawiki/2020/0/0c/T--IISER_Berhampur--_exp5.png)

**Figure 3b** Virtual Gel Images (1 kb Marker, 1% Agarose gel

### 3. In Silico Digest for excising mTurquiose

Restriction Enzymes: MfeI and AgeI

Length: 711 bp, 26.7 kDa protein, 237 amino acids

### 4. In Silico Digest for excising cp173Venus

Restriction Enzymes: KpnI and NotI

Length: 735 bp, 27.3 kDa protein, 245 amino acids

![](https://static.igem.org/mediawiki/2020/b/bd/T--IISER_Berhampur--_exp6.png)

**Figure 4a** Circular Digests and ORFs generated

![](https://static.igem.org/mediawiki/2020/f/f1/T--IISER_Berhampur--_exp7.png)

**Figure 4b** Virtual Gel Images (1 kb Marker, 1% Agarose gel)

# Competent Cell Preparation

We planned to do this in the last week of October. But with the current availability of competent cells in the department, the competent cells were acquired from the stock which was stored at -80°C. DH5α is the available competent cell which was prepared in the month of March.

* Inoue transformation buffer prepared and filter sterilized using a 0.45 mm Nalgene filter. This was chilled on ice.
* A single DH5α colony from a streaked plate was inoculated into 25 ml of LB medium in 250 ml flask. This culture flask was incubated at 37 °C overnight, with shaking at 250 RPM.
* After approximately 8 hours of starter culture growth, three 1 litre flasks containing 250ml of LB were inoculated with differing volumes of the starter culture as follows, 2 ml, 5 ml and 10 ml respectively.
* The flasks were incubated at 22 °C at 100 RPM, overnight.
* After overnight incubation, the OD was measured for 1 ml of the culture, measured at OD600.
* The 10 ml starter culture inoculated flask crossed the OD and hence it was discarded.
* The 5 ml starter culture inoculated flask reached the OD in about 4 hours, this was immediately placed in ice bucket for 10 minutes.
* The cells were harvested by centrifugation in the Eppendorf centrifuge at 3000 rpm for 10 minutes at 4 °C.
* After discarding the supernatant in disinfectant containing disposal flask, the pellet was resuspended in 80 ml of ice cold Inoue transformation buffer. The resuspension was as gentle as possible with minimum pipetting and then mixed by swirling.
* The cells were harvested by centrifugation at 3000 RPM for 10 minutes at 4 °C.
* The supernatant was discarded as described above.
* The cell pellet was carefully resuspended in 20 ml of ice-cold Inoue transformation buffer. To this mix, 1.5 ml of DMSO was added. This was mixed gently, and 400 µl aliquots were made into prechilled 1.5ml tubes.
* Once the aliquoting was done, the competent cells efficiency was tested by using different controls.
* The competent cell aliquots were stored at -80 °C.

# Bacterial Transformation

In order to carry out the transformation, we started with the preparation of antibiotics followed by the preparation of fresh media and agar plates.

**Antibiotic**: The antibiotic used for this transformation was ampicillin (Himedia CMS64) which was procured from Dr. Vinay’s research lab and prepared a stock solution with a concentration of 100 mg/mL. The working concentration was made by diluting the stock with Double Distilled Water (DDW) to 100 µg/mL.

**LB media preparation**: Luria Bertani Broth, Miller (Himedia M1245) media was prepared for a total volume of 200 mL with 5 gm of Miller. With a thorough mixing of solution, media was transferred to a 500 mL air tightened conical flask for autoclaving

**LB-Amp Agar plate preparation**: For the preparation of four agar plates a composition of Luria Bertani Agar media (Himedia M1151) was prepared with a total volume of 120 mL with 4.8 gm of agar. The autoclaving proceeded for 45 minutes and ampicillin was added to the media after cooling. The media was now enriched with antibiotics and WE proceeded to make plates.

As the plates and media were made, for proceeding to bacterial transformation, 200 µL of DH5α and 1 µL DNA of plasmid pCKI were taken in a 1.5 mL eppendorf tube and mixed, incubated on ice for 30 minutes. This was followed by heat shock at 42°C for 90 seconds and immediately incubated on ice for 5 minutes; this heat shock will allow the integration of DNA to the E.coli competent cells and the final incubation on ice will help to seal this cell part respectively. 300 µL of LB was added and incubated for 40 minutes at 37°C in shaking condition. With the completion of this step the cells were centrifuged, the pellet was resuspended in 100 µL of LB. Now, the cells were plated on LB-Amp plates (see above for the preparation protocol) uniformly using a sterilized spreader and left the plate inverted at 37°C for 12 hours in an incubator with air fan mode on. A strict regime of 12 hours was maintained to avoid the growth of any satellite colonies.

## Bacterial Culture for Plasmid Preparation

With the completion of 12 hour incubation, we could observe colonies in the LB-Amp plates. A single colony was picked up using a pipette tip and inserted in a falcon tube with 5 mL LB media added with Ampicillin (5 µL). Replicates were made and left for shaking incubation with an RPM of 100 at 37°C for 12 hours.

## Plasmid Preparation

* The 5ml culture was centrifuged in batches of 1.5 ml, three times in 1.5 ml tubes.
* The pelleted ~4ml culture was resuspended in 600 µl of TE buffer.
* The resuspended solution was mixed with 100 µl of 7X lysis buffer and mixed by inverting the tube.
* The solution changed from opaque to clear blue, which is a mark of complete lysis.
* Immediately, 350 µl of cold neutralization buffer was added and mixed by inverting, the solution turned yellow in colour.
* The above sample was centrifuged at 13000 RPM for 3 minutes.
* The supernatant was carefully transferred to the Zymo spin column and centrifuged for 13000 rpm for 15 seconds.
* The flow through was discarded.
* The column was washed with 200 µl of endo-wash buffer. The tube was centrifuged at 13000 rpm for 15 seconds.
* The column was washed with a 400 µl wash buffer at 13000 rpm for 30 second.
* The column was transferred from the collection tube to a 1.5 ml microcentrifuge tube.
* 30 µl elution buffer was added to the column. After incubation at room temperature for 1 minute, the column was centrifuge again at 13000 rpm for 15 second and DNA was eluted.

## Concentration estimation by nanodrop (Thermofisher)

2 µl of the plasmid DNA was used for concentration estimation

Replicate 1: 70.6 ng/ µl

Replicate 2: 93.7 ng/ µl

# RE Digestion

| COMPONENTS (µL) | RESTRICTION DIGESTION 1 (EcoRI) | RESTRICTION DIGESTION 2 (HindIII) | RESTRICTION DIGESTION 3 (EcoRI/HindIII) |
| --- | --- | --- | --- |
| PLASMID PCKI | 3.5 (from 70 ng/µL conc. ) | 3.5 (from 70 ng/µL conc. ) | 2.6 (from 95 ng/µL conc.) |
| &10X BUFFER | 2.5 (1X conc.) | 2.5 (1X conc.) | 2.5 (1X conc.) |
| ENZYME 1 (EcoRI) | 0.5 |  | 0.5 |
| ENZYME 2 (HindIII) |  | 0.5 | 0.5 |
| NUCLEASE FREE WATER | 18.5 | 18.5 | 18.9 |
| TOTAL (µL) | 25 | 25 | 25 |

- Concentration estimation by nano-drop

**Incubation conditions**: Water bath at 37℃ for 1 hour.

# Agarose Gel Electrophoresis

The fragments of the plasmid after single and double digestions were run on an agarose gel to visualise and analyse the DNA.

**Protocol**

* 0.8% agarose gel was prepared by dissolving 0.4 g of agarose powder (HiMedia, MB002-100G) in 50 mL TBE buffer (of 1X concentration) in a microwavable flask.
* 12.5 µL of EtBr (final conc. = 0.5 µg/mL) was added and mixed properly after the solution cooled down to a tolerable temperature.
* The agarose solution was poured into a casting tray with an 8-well comb in place.
* The solution was allowed to cool and solidify at room temperature.
* The gel was moved into the gel box and 1X TBE buffer was poured into the box such that the gel got immersed completely.
* 25 µL of each sample and 5 µL loading dye were mixed for loading into each well.
* 20 µL of each sample was loaded onto each well in the sequence as shown in Fig.5. Two ladders were used; Purple 1kb plus ladder (NEB - N055OS) and a ladder of 100bp - 3kb (HiMedia).
* The gel was run at 120 V for around 1.5 hours.
* The bands were visualised under UV light in the Gel Doc imaging system (UVP ChemStudio - Analytik Jena).

![](https://static.igem.org/mediawiki/2020/c/c5/T--IISER_Berhampur--_eng1122.png)

**Figure 5:** Restriction digestion of pCKI with EcoRI and HindIII, 0.8% agarose gel, 1X TBE

## Results

In the 1st and 6th well, we can observe the ladders, the first one of which is of a higher molecular weight (1kb above) ladder and the other one is a broad-ranging ladder from 100 base pair to 300-kilo base pair. As we used the uncut plasmid having a size of 7 kb, the first ladder is suitable to confirm the size and the second ladder for confirming low molecular weight fragments generated upon RE digestion. Hence, the use of both ladders will help to locate the specific fragments.

The second well contains an uncut (i.e., no RE added) plasmid and as the position of the band is not too far from the well, this possibly depicts the actual size of the plasmid which is ~7kb. Ideally, however, a denser band should have been observed, but in this case as the concentration of plasmid used was 250 ng, so the brightness in this picture in a way, mirrors the concentration of plasmid.

In the third well (digested with the RE EcoRI), and fourth wells (digested with the RE HindIII), a comparison with the in silico digests, reveals that we should obtain one band (i.e., 1 fragment) of size 7 kb since both these enzymes have a single site in the sequence (Refer to Figure 3a). This situation is not reproduced experimentally however and we could observe two bands in both these lanes and fragments were not in the exact expected size range.

In the combination of EcoRI and HindIII, which is a double digest and in reference with the in silico analysis we should have observed 2 bands (6.6 kb + 0.4kb) but, only a single clear band is visible here, which means the double digest shows only linearization suggesting incomplete digestion. The main conclusion is that the Restriction Enzymes digestion needs to be repeated with increased/different enzymes and additional incubation time. Also for the double digest, the compatibility of the buffer for both enzymes remains to be confirmed. Given that these are our optimization experiments, we will use these to troubleshoot and modify the protocols for the experiments using our FRaPPe constructs.

# Mammalian Cell Culture

Since our entire project runs around HEK 293 cells, this SOP is to elaborate on the principles and protocols for maintaining HEK 293 cells in culture and preparation for transfection.

# HEK Cell Line

The Human Embryonic kidney 293 cells are specific cell line originally derived from human embryonic kidney cells grown in tissue culture.

HEK 293 cells were generated in 1973 by transfection of cultures of normal human embryonic kidney cells with sheared adenovirus 5 DNA in Alex van der Eb’s laboratory in Leiden, the Netherlands.

There is evidence that HEK 293 cells and other human cell lines generated by adenovirus transformation of human embryonic kidney cells have many properties of immature neurons, suggesting that the adenovirus preferentially transformed the neuronal lineage cell of the original kidney culture.

HEK 293 cells have a complex karyotype, exhibiting two or more copies of each chromosome and with a modal chromosome number 64. They are described as hypotriploid, containing less than three times the number of chromosomes of a haploid human gamete. (Chromosomal abnormalities include three copies of X chromosomes and four copies of chromosome 17 and chromosome 22).

## Purpose of choosing HEK 293

It is easy to grow and transfect cells and has been widely used for cell biology industry to produce therapeutic proteins and viruses for gene therapy. It is also not a cancerous cell line unlike Hela.

High reproducibility, which makes it preferable over other less-robust and slow-growing cell lines.

# Cell Culture Work Flow

## ISOLATE

* Separate fresh tissue of interest.
* Treat tissue with enzyme(s) (e.g. trypsin, collagenase, protease) and/or mechanically isolate cells.
* Wash, count and seed cells.

## VERIFY

* Examine cells under a brightfield microscope to access their growth state, attachment to culture vessels/flasks and to check for any signs of infection.
* Monitor cells for the following days until they reach confluence.
* Verify isolated cell types by their morphology and expressed biomarkers.

## CULTURE

* Passage cells to propagate cell line.
* Make master and working cell banks.
* Immortalize cells if necessary.

## INVESTIGATE

* Plan and execute experiments.
* Keep monitoring cell state and possible infections using a brightfield microscope.

# Basic cell culture equipment

The specific equipment of a cell culture laboratory depends on the type of research conducted. However, all cell culture laboratories have the same common equipment, free from pathogenic microorganisms

* Wrapped disposable pipettes
* Waste container
* Glass pipette (if using)
* Waste liquid
* Reagents and media
* Pipettor
* Work surface
* 70% ethanol antiseptic
* Autoclave

# Reagents

* Medium: 500ml Dulbecco’s modified Eagle Medium **(SIGMA, D5796 ; HIMEDIA, TC185)**, FBS **(HYCLONE, SV30160.03)**, Penicillin Streptomycin solution **(HIMEDIA, A0002)**
* Trypsin **(SIGMA, 59428C)**
* Cell culture 6 well plate – equivalent to 30 mm plates **(TARSONS, 950040)**
* Cryovials (TARSONS, 523182)
* Cell culture vented flasks with slight inclinatioN **(T25 TARSONS, 523182)**

# Apparatus

5 % CO2 incubator, hemocytometer, inverted microscopes (to observe confluent cells, light source from below)

# Procedure

* The media was discarded from vented flasks (prewarm the media at 37°C).
* The medium was aspirated from the flask and wash with PBS (2 ml) to remove all traces of media, serum etc. so that trypsin can act.
* It was gently swirled to mix. Some cells may detach by the force already.
* Next, 1 ml of trypsin was added and swirled gently.
* Incubated at 37°C for 2 minutes.
* One flask was seeded for 1 F, one made into stock, others packaged.
* Media was to neutralize trypsin (9 ml media + 1 ml trypsin).
* If thread-like structure appears, which indicates cells are in clumps. (Note: Thread-like structures before step 1 may indicate fungal fungal spore contamination).
* Mixture was pipetted up and down to homogenize. (No bubble formation)

## STEP 1 : Contribute to ROS

* Aliquots taken for cell count (for seeding for 1F)
* 3 coverslips were put in each 30mm plate of 6 well-plate, 2.5 ml (approx.) media was added into each plate (maximum can be 3 ml) + 0.5 ml cells in each plate.

## STEP 2 : Passaging of cells

* For passage, solutions were distributed and 4 ml media added into new culture flasks and cells washed with PBS and trypsinized at 37°C.

## STEP 3 : Stock preparation

* Centrifuged after aliquoting -->freezing media + DMSO and serum -->stored at - 80°C for 3-4 weeks

## Maintaining HEK 293 cells in culture

HEK 293 cells should be grown in a monolayer, preferably in plastic petri dishes or flasks. Under optimum growth conditions (37°C, 5% co2), 293 cells double after every 36hr. To maintain consistency, one should not passage cells indefinitely. For best results, one may use low passage 293 cells for transfection and titration procedures.

To prevent contamination, work with media and uninfected cells should be done in a vertical laminar flow hood, using sterile technique. Ideally, we should use another hood for all virus work. All virus-contaminated materials, including fluids, must be autoclaved or disinfected with 10% bleach or a chemical disinfectant before disposal.

## Antiseptic techniques required while working with cell culture

To have a successful cell culture, it is essential to maintain a contamination free environment (bacteria, fungi, etc.). Aseptic techniques ensure that no microorganisms enter the cell culture set-ups. Cell culture sterility is ensured by a defined set of procedures.

| HANDLING | REAGENTS/MEDIA | WORKPLACE |
| --- | --- | --- |
| Slow/careful handling | Pre-sterilization of all reagents/equipment | Cell culture hood should function properly |
| Sterilization of all items before starting. | No contamination in reagents (expiration date, appearance normal). | Frequent de-contamination (hood, fridge etc.) |
| No touching of sterile items to non-sterilized surfaces |  | Work area should be sterile and tidy. |

# Electroporation

Electroporation— the use of high-voltage electric shocks to introduce DNA into cells—is a procedure that is gaining popularity for standard gene transfer. It also allows the generation of genetically modified mice. It can be used with most cell types, yields a high frequency of both stable transformation and transient gene expression, and, because it requires fewer steps, can be easier than alternate techniques.

The method uses an electric field that transiently permeabilizes the membrane. During this period the foreign material can enter the cell. This method-

* Works well with almost all cell types.
* Ensures high transfection efficiency.
* Requires optimization as cell permeability can lead to cell damage.
* Experienced user needed.
* Quite an expensive method.

## Materials

Mammalian cells to be transfected.

Complete medium without and with appropriate selective agents

Electroporation buffer, ice-cold

Linear or supercoiled, purified DNA preparation

Beckman JS-4.2 rotor or equivalent

Electroporation cuvettes (Bio-Rad #165-2088) and power source

Additional reagents and equipment for stable transformation in selective medium and for harvesting transfected cells

## Procedure

* Cells to be transfected are grown to late-log phase in complete medium. Each permanent transfection will usually require 5 × 106 cells to yield a reasonable number of transfectants. Each transient expression may require 1–4 × 107 cells, depending on the promoter.
* Cells are then harvested by centrifuging 5 min at 640 × g (1500 rpm in a JS-4.2 rotor), 4°C. Adherent cells are first trypsinized and the trypsin inactivated with serum.
* Cell pellet is resuspended in half its original volume of ice-cold electroporation buffer. The choice of electroporation buffer may depend on the cell line used.
* Again, cells are harvested by centrifuging for 5 min as in step 2.
* 1ml aliquots are dispensed into labelled freezing vials and place in a cell freezing container (reduces temperature ~1°C/min) at -80°C overnight.
* Cells are resuspended at 1 × 107/ml in the electroporation buffer at 0°C for permanent transfection. Higher concentrations of cells (up to 8 × 107) may be used for transient expression.
* 0.5-ml aliquots of the cell suspension are transferred into desired number of electroporation cuvettes set on ice.
* DNA added and electroporation of the cells done.
* DNA added to cell suspension in the cuvettes on ice.
* DNA/cell suspension is mixed by holding the cuvette on the two “window sides” and flicking the bottom. Incubated for 5 min on ice.
* The cuvette is placed in the holder in the electroporation apparatus (at room temperature) and shocked one or more times at the desired voltage and capacitance settings. The number of shocks and the voltage and capacitance settings will vary depending on the cell type and should be optimized.
* After electroporation, the cuvette containing cells and DNA is returned to ice for 10 min.
* Transfected cells are diluted 20-fold in nonselective complete medium and cuvette rinsed with this same medium to remove all transfected cells.
* Cells are responded at 1 × 107ml in the electroporation buffer at 0°C for permanent transfection. Higher concentrations of cells (up to 8 × 107) may be used for transient expression.
* For stable transformation: Cells should be grown for 48 hours (about two generations) in nonselective medium, then transferred to antibiotic-containing medium. Selection conditions will vary with cell type.
* For transient expression: Cells should be incubated for 50 to 60 hours, then harvested for transient expression assays. Transfected cells can be visualized by standard transient expression assays.

# Wet Lab Notebook

**Day 1 – 17/10/2020**

pCKI plasmid was kindly provided by Dr. Vinay Bulusu.

Agar media and plates were prepared and ampicillin was spread over the plate.

Transformation of the pCKI plasmid and DH5α competent cells was performed.

Transformed cells were plated on LB-ampicillin agar plates along with an additional control plate for the competent cells. These plates were incubated at 37°C for 12 hours.

**Day 2 – 18/10/2020**

There were few colonies on the plates, which suggested that the transformation process had not gone well. So, we decided to repeat the process.

This could be because of problems with the cells or the culture media.

The LB media and LB-Amp agar plates were prepared freshly. After transformation of the plasmid and competent cells, the cells were plated and incubated at 37°C for 12 hours.

We used two different plasmid concentrations.

**Day 3 – 19/10/2020**

With the completion of 12 hours incubation, we were successful in witnessing the colonies in both plates. Stored these plates at 4°C to proceed further.

A single colony was added to a falcon tube containing 5 mL of LB Broth media with different concentrations of ampicillin and two replicates were taken for each concentration. A pipette tip was used to scoop out a single colony.

The falcon tubes were placed in a shaking incubator with 100 RPM for 12 hours at 37°C.

**Day 4 – 20/10/2020**

We were able to observe the dense colour of the media signifying the growth.

Cells were processed for plasmid extraction.

Plasmids that were extracted, were stored at -20 °C

**Day 5 - 22/10/20**

Restriction digestion of the plasmid was done to confirm the plasmid.

Single enzyme digests and Double enzyme digests using 250ng of plasmid were performed at 37°C for 1 hour.

**Day 6 - 23/10/20**

Restriction digested plasmids were subjected to electrophoresis on 0.8% agarose gel.

Reagents for cloning and plasmid purification were received from NEB.

**Day 7 - 24/10/20**

Cell culture media was prepared and incubated in the cell culture incubator for sterility check.

**Day 8 - 25/10/20**

HEK293 cells were recovered from previously cryopreserved vials.

Plasmid restriction digestion was repeated with 1µg of pCKI

# References

1. Bulusu V et al., (2017) Spatiotemporal Analysis of a Glycolytic Activity Gradient Linked to Mouse Embryo Mesoderm Development. Dev Cell. 40(4):331-341.

2. Klarenbeek, J. et al., (2015). Fourth-generation epac-based FRET sensors for cAMP feature exceptional brightness, photostability and dynamic range: characterization of dedicated sensors for FLIM, for ratiometry and with high affinity. PloS one, 10(4), e0122513. https://doi.org/10.1371/journal.pone.0122513/

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# Team:IISER Berhampur/Results

IISER-BPR IGEM

[Home](https://2020.igem.org/Team%3AIISER_Berhampur)

Project▼

[Overview](https://2020.igem.org/Team%3AIISER_Berhampur/Project/Overview)
[Description](https://2020.igem.org/Team%3AIISER_Berhampur/Description)
[Implementation](https://2020.igem.org/Team%3AIISER_Berhampur/Implementation)
[Experiments](https://2020.igem.org/Team%3AIISER_Berhampur/Experiments)
[Engineering](https://2020.igem.org/Team%3AIISER_Berhampur/Engineering)
[Notebook](https://2020.igem.org/Team%3AIISER_Berhampur/Project/Notebook)
[Contribution](https://2020.igem.org/Team%3AIISER_Berhampur/Contribution)

[Modelling](https://2020.igem.org/Team%3AIISER_Berhampur/Model)

Team▼

[Members](https://2020.igem.org/Team%3AIISER_Berhampur/Team)
[Attributions](https://2020.igem.org/Team%3AIISER_Berhampur/Attributions)
[Collaborations](https://2020.igem.org/Team%3AIISER_Berhampur/Collaborations)

[Parts](https://2020.igem.org/Team%3AIISER_Berhampur/Parts)
[Human Practices](https://2020.igem.org/Team%3AIISER_Berhampur/Human_Practices)
[Science Communication](https://2020.igem.org/Team%3AIISER_Berhampur/Education)
[Splash](https://2020.igem.org/Team%3AIISER_Berhampur/Splash)
[Awards](https://2020.igem.org/Team%3AIISER_Berhampur/Awards)
[Graphics](https://2020.igem.org/Team%3AIISER_Berhampur/Graphics)

![](https://static.igem.org/mediawiki/2020/6/69/T--IISER_Berhampur--gold.png)

# Results

You can describe the results of your project and your future plans here.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)