Team:IISER Bhopal/Engineering

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Engineering Success

Introduction

Our kill switch characterization demonstrates our engineering success and the results for the same are presented in the results. You can also view the description, mechanism, modelling and results of the kill switch in the registry part page - Part:BBa_K3848004.

Unfortunately due to the COVID-19 pandemic, we had delayed lab access and inconsistent deliveries by the vendors causing our core project lab work to begin in September, hence we were not able to execute all of our modules. But we were successfully able to demonstrate the engineering success of our kill switch.

All the protocols mentioned here are described in the protocols page, here we present the workflow of the project. In the workflow, protocols described in the protocols page are highlighted.

MODULE 1

Initial Characterization:-

Bacterial Growth Kinetics ‎‎‎‎‎‎‎ ‎‎‎

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Bacterial Growth Kinetics

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For competent cell preparation, we used the Hanahan or Calcium chloride method. DH5𝞪 cells were made competent. We chose DH5𝛂 over BL21 on the suggestion of Dr. Vikas Jain of the Department of Biological Sciences of IISER Bhopal, he told us that plasmids are less stable in BL21(DE3) than DH5𝛂 and DH5𝛂 can also express proteins. We also checked the efficiency. We then generated ultracompetent cells from competent cells.
Transformation of bacteria with pUC 19, pMB1

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Transformation of bacteria with pUC 19, pMB1

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pUC and pMB1 both were used because they have similar ori and pMB1 itself codes for GFP and our AND gate reporter construct is also GFP based, hence it cannot be cloned in pMB1. Hence we used pUC19 to express the reporter.

Extraction of Plasmid and Checking for plasmids pMB1

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Extraction of Plasmid and Checking for plasmids pMB1 , pUC (Restriction Digest)

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Used the Promega plasmid extraction kit. Restriction digestion also done using Promega kit. Then we ran the products in agarose gel electrophoresis to confirm the existence of the right plasmid in the cells.

PCR To amplify the ordered gene constructs

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PCR To amplify the ordered gene constructs

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Our initial order from IDT was hugely delayed by two months due to the pandemic, hence we then decided to order from Twist Biosciences. For that we had to generate new restriction sites, and divide our full cassette into 5 parts (excluding the reporter construct) that were to be ligated later on. We used the adapter sequences from the gene fragments ordered from Twist Biosciences to amplify our individual gene fragments. PCR protocol from Promega was followed.

MODULE 2

Cloning & Expression

Restriction Digestion ‎‎‎‎‎‎‎ ‎‎‎

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Restriction Digestion

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Since our full cassette was now divided into 5 parts, we had to restrict and ligate individual parts sequentially.
  • Kill switch cassette restriction reactions:- SalI - Antiholin - PstI PstI - Holin - XhoI SalI - pMB1 - XhoI (plasmid double digest) We could not ligate lldR and lldP to the kill switch cassette because of inconsistent restriction sites in the gene fragments and the plasmid. But, in spite of this, we used the host lldP, lldR genes for the kill switch regulation and tested whether it behaves similarly to a plasmid encoded lldPR. And the host lldPR also worked and showed graded response in different concentrations of lactate (see results page)
  • Secretion cassette restriction reactions:- EcoRI - lldP - XbaI XbaI - lldR - HindIII HindIII - Trail-Smac fusion construct - SalI EcoRI - pMB1 - SalI (plasmid double digest)
  • Reporter cassette restriction reactions:- EcoRI - lldP - XbaI XbaI - lldR - HindIII HindIII - Trail-Smac reporter construct - SalI EcoRI - pUC - SalI (plasmid double digest) We cloned the reporter construct in pUC19 because our reporter is an eGFP construct and pMB1 has intrinsic GFP expression.
    • Sequential ligation

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    Sequential ligation

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    Sequential ligation of the 5 parts in which our constructs arrived, and we anticipate significant reduction in ligation efficiency after each ligation. For troubleshooting, we plan to use overlapping PCR by ordering new primers for the same.

    Transformation, Selection

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    Transformation, Selection

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    Antibiotics, Blue-white selection

    AND Gate Characterization

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    AND Gate Characterization

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    GFP fluorescence measurement (for reporter) and the isolation of the secreted fusion peptide by Ni-NTA chromatography.

    Kill Switch Characterization

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    Kill Switch Characterization

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    Kill Switch Characterization: We initially started off choosing quorum sensing as the basis of the kill switch, but upon some brainstorming, we concluded that this type of kill switch can potentially lead to systemic mode of signaling to various harmful bacteria as well, and hence we scrapped that plan. Then we decided to use a lactate based kill switch which would be calibrated with the lactate concentrations found in the tumor microenvironment (see results). Further, after consulting Dr. Vikas Jain of the Department of Biological Sciences of IISER Bhopal, we also included the lambda repressor in the kill switch so as to allow the bacteria enough time after IV injection to localize into the tumor, which was not possible earlier because the kill switch could be triggered as soon as the bacteria is injected into the body. We characterized the kill switch by CFU measurements in different lactate concentrations.

    TRAIL-Smac FP expression

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    TRAIL-Smac FP expression

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    Protein Purification & Quantification - Isolation of the secreted fusion protein by Ni NTA chromatography, Spectrophotometry (280nm) , Bradford Assay followed by SDS PAGE(Shakti)

    MODULE 3

    Cell Line Studies

    Cell line Culture ‎‎‎‎‎‎‎ ‎‎‎

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    Cell line Culture

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    MCF7 or T47D ,NCI-H460, A549 ,SK-HEP-1 cancer cell lines.

    TRAIL Binding Assay

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    TRAIL Binding Assay

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    In this assay, we first saturate TRAIL receptors, then introduce TRAIL-Smac fusion peptide and check the presence of TRAIL. Then we do a follow-up experiment where we introduce the fusion peptide only to a new cell culture and check whether the fusion peptide is bound or not.

    Cleavage of Fusion Protein

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    Cleavage of Fusion Protein

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    We treat our fusion protein with MMP-3/9 and uPA proteases which are overexpressed in cancer cells and check for the cleavage of the fusion peptide via SDS-PAGE. 2 bands would be found, one of high molecular weight (uncleaved protein) and lower molecular weight (cleaved protein). We will use western blotting to detect the proteins.

    Internalization of Smac

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    Internalization of Smac

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    We check Smac internalization after treating with the fusion peptide, by intracellular immunostaining of Smac.

    Apoptosis Detection Assays

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    Apoptosis Detection Assays

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    MTS Assay and Annexin-V, pI staining - Flow Cytometry were used to detect apoptosis in cancer cells introduced to the toxicant (TRAIL-Smac fusion protein).

    Mechanism of Apoptosis

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    Mechanism of Apoptosis

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    Caspase Activation Assays:- Caspase-8 activation assay Caspase-3/7 activation assay DNA Fragmentation assay:- Tunel Assay followed by gel electrophoresis of isolated DNA

    MODULE 4

    Future Aspects

    B. longum studies ‎‎‎‎‎‎‎ ‎‎‎

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    B. longum studies

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    Growing B. longum in anaerobic conditions Optimizing our constructs for B, longum. We need not change the plasmid as pMB1 can operate in both E. coli and B. longum. Making electrocompetent B. longum and electrotransformation of B. longum.

    Animal Model Studies

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    Animal Model Studies

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    For in vivo studies, we plan to introduce cultured B. longum intravenously in PDX (patient derived xenograft) mouse models. And then check the remission or regression of solid tumor size. For dosage information and localization of B. longum into tumor tissue refer:- Sasaki T, Fujimori M, Hamaji Y, Hama Y, Ito K, Amano J, Taniguchi S. Genetically engineered Bifidobacterium longum for tumor-targeting enzyme-prodrug therapy of autochthonous mammary tumors in rats. Cancer Sci. 2006 Jul;97(7):649-57. doi: 10.1111/j.1349-7006.2006.00221.x. Erratum in: Cancer Sci. 2006 Sep;97(9):961. PMID: 16827806. Protocols for handling of mice and generation of PDX mice are given in the protocols page.

    Module 5

    Proposed implementation

    Our product Trasmax to be administered as IV injection is a pharmaceutical product and our end-users are cancer patients. Our target group mainly includes early stage and pre- metastasis stage cancer patients.

    This novel treatment therapy can be used to treat cancer patients at a much cheaper cost with minimal side-effects. Though we do not have accurate wet-lab data to support this claim, as per our understanding from previous literature reviews, B.longum elicited minimal immune response when tested on Guinea pigs.

    We have completed a thorough analysis of the market (how we intend to reach and diversify our market), competitors, customer needs, requirements (the assistance we can give to the diabetes patients), patient demographics etc. These are just some of the various aspects we have clearly identified and explained in the entrepreneurship section of Implementation. Our safety policies are extremely stringent which will not only ensure the safety of the patient but also that of the environment. We have rigorously tested for biosafety issues and tried to eliminate them in the design stages.