iGEM 2021 — Team IISER_Bhopal

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# Team:IISER Bhopal/Notebook

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# Notebook

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# 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.](http://parts.igem.org/wiki/index.php?title=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:-

![](https://static.igem.org/mediawiki/2021/e/e6/T--IISER_Bhopal--engineering1.png)

###### Bacterial Growth Kinetics ‎‎‎‎‎‎‎ ‎‎‎

[More](#popup1)

## Bacterial Growth Kinetics

[×](#idhar)

**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.**

![](https://static.igem.org/mediawiki/2021/8/85/T--IISER_Bhopal--engineering2.png)

###### Transformation of bacteria with pUC 19, pMB1

[More](#popup2)

## Transformation of bacteria with pUC 19, pMB1

[×](#idhar)

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.

![](https://static.igem.org/mediawiki/2021/8/8f/T--IISER_Bhopal--engineering3.png)

###### Extraction of Plasmid and Checking for plasmids pMB1

[More](#popup3)

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

[×](#idhar)

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.

![](https://static.igem.org/mediawiki/2021/6/6a/T--IISER_Bhopal--engineering4.png)

###### PCR To amplify the ordered gene constructs

[More](#popup4)

## PCR To amplify the ordered gene constructs

[×](#idhar)

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

![](https://static.igem.org/mediawiki/2021/4/40/T--IISER_Bhopal--engineering5.png)

###### Restriction Digestion ‎‎‎‎‎‎‎ ‎‎‎

[More](#popup5)

## Restriction Digestion

[×](#udhar)

**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.**

![](https://static.igem.org/mediawiki/2021/0/07/T--IISER_Bhopal--engineering6.png)

###### Sequential ligation

[More](#popup6)

## Sequential ligation

[×](#udhar)

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.

![](https://static.igem.org/mediawiki/2021/7/7b/T--IISER_Bhopal--engineering7.png)

###### Transformation, Selection

[More](#popup7)

## Transformation, Selection

[×](#udhar)

Antibiotics, Blue-white selection

![](https://static.igem.org/mediawiki/2021/c/cd/T--IISER_Bhopal--engineering8.png)

###### AND Gate Characterization

[More](#popup8)

## AND Gate Characterization

[×](#udhar)

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

![](https://static.igem.org/mediawiki/2021/2/2e/T--IISER_Bhopal--engineering9.png)

###### Kill Switch Characterization

[More](#popup9)

## Kill Switch Characterization

[×](#udhar)

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.

![](https://static.igem.org/mediawiki/2021/f/f1/T--IISER_Bhopal--engineering10.png)

###### TRAIL-Smac FP expression

[More](#popup10)

## TRAIL-Smac FP expression

[×](#udhar)

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

![](https://static.igem.org/mediawiki/2021/4/4c/T--IISER_Bhopal--engineering11.png)

###### Cell line Culture ‎‎‎‎‎‎‎ ‎‎‎

[More](#popup11)

## Cell line Culture

[×](#kidar)

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

![](https://static.igem.org/mediawiki/2021/f/f3/T--IISER_Bhopal--engineering12.png)

###### TRAIL Binding Assay

[More](#popup12)

## TRAIL Binding Assay

[×](#kidar)

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.

![](https://static.igem.org/mediawiki/2021/7/7c/T--IISER_Bhopal--engineering13.png)

###### Cleavage of Fusion Protein

[More](#popup13)

## Cleavage of Fusion Protein

[×](#kidar)

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.

![](https://static.igem.org/mediawiki/2021/7/7c/T--IISER_Bhopal--engineering14.png)

###### Internalization of Smac

[More](#popup14)

## Internalization of Smac

[×](#kidar)

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

![](https://static.igem.org/mediawiki/2021/7/7c/T--IISER_Bhopal--engineering16.png)

###### Apoptosis Detection Assays

[More](#popup14)

## Apoptosis Detection Assays

[×](#kidar)

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).

![](https://static.igem.org/mediawiki/2021/7/7c/T--IISER_Bhopal--engineering16.png)

###### Mechanism of Apoptosis

[More](#popup15)

## Mechanism of Apoptosis

[×](#kidar)

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

![](https://static.igem.org/mediawiki/2021/f/fa/T--IISER_Bhopal--logo.png)

###### B. longum studies ‎‎‎‎‎‎‎ ‎‎‎

[More](#popup20)

## B. longum studies

[×](#kedar)

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.

![](https://static.igem.org/mediawiki/2021/f/fa/T--IISER_Bhopal--logo.png)

###### Animal Model Studies

[More](#popup21)

## Animal Model Studies

[×](#kedar)

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.

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:IISER Bhopal/Experiments

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:IISER Bhopal/Results

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# Results

## Wetlab Results

Due to Covid-19 restrictions, we had delayed lab access. We started our main lab work by the end of September 2021, when our ordered gene constructs arrived. So, we have only done the characterization of the Holin-Antiholin Kill Switch from the lab. Although we were successful in inserting other gene cassettes into E. Coli, we were unable to do its characterization and the cell line studies due to time limitations.

### Preparation of Competent and & Ultracompetent E.Coli

#### Competent DH5-α & BL-21(DE3)-By Hanahan method (CaCl2)

![](https://static.igem.org/mediawiki/2021/f/f6/T--IISER_Bhopal--Engineering_Success--Wet_Lab_Results--image_1.jpg)

![](https://static.igem.org/mediawiki/2021/c/cd/T--IISER_Bhopal--Engineering_Success--Wet_Lab_Results--image_3.jpg)

*Fig. 2: i) BL-21(DE3) competent cells efficiency check by transformation with pUC19, plated in 100µg/mL Ampicillin.*

### Ultracompetent DH5-α by Inoue Method

![](https://static.igem.org/mediawiki/2021/2/21/T--IISER_Bhopal--Engineering_Success--Wet_Lab_Results--image_4.jpg)

*Fig.3: Ultracompetent DH5-α check-i)Ultracompetent DH5𝛂 transformed with 100 ng pUC19, plated in 100 µg/ml Ampicillin Plates
ii)Transformation with 100 pg pUC19, plated in 100µg/ml Ampicillin Plates iii)No Antibiotic Control (No pUC19).*

### PCR, RESTRICTION DIGESTION & LIGATION OF THE CONSTRUCTS

#### 1) PCR Amplification of Constructs

![](https://static.igem.org/mediawiki/2021/c/cf/T--IISER_Bhopal--Engineering_Success--Wet_Lab_Results--image_5.jpg)

*Fig. 4:Gel Electrophoresis image of the PCR Amplified Gene constructs. From Right: i)1 kb ladder ii) TRAIL-Smac Fusion Construct iii)AND Gate Reporter iv)LldP
v)LldR vi)Antiholin construct vii)Holin viii)1 kb ladder*

#### 2) Restriction digestion of the Constructs

![](https://static.igem.org/mediawiki/2021/6/66/T--IISER_Bhopal--Engineering_Success--Wet_Lab_Results--image_6.jpg)

*Fig. 5:Restriction digestion of each construct (From left to right)-i)pUC19 plasmid with pst1 and Xba 1 (2.6kb) ii)Trail-Smac fusion peptide with pst1 and xba1 (1.3kb) iii)LldP construct by Xba1 (1.7 kb) iv)AND Gate Reporter construct with pst1 and xba1 (1.3 kb)
v)LldR construct by Xba1 (0.9 kb) vi)Antiholin-Lamda Repressor construct by Pst1 (1.4kb) vii) Holin-Endolysin construct: by Pst1 (1.4 kb)*

#### 3) Ligation of each unit

![](https://static.igem.org/mediawiki/2021/9/9e/T--IISER_Bhopal--Engineering_Success--Wet_Lab_Results--image_7.jpg)

*Fig. 6:From left to right- **i)TRAIL-Smac fusion protein**:Ligation of TRAIL-Smac FP (1.3kb) into pUC 19 plasmid (2.6kb ) [D+pUC].3.9kb ligated band is visible. **ii)AND Gate**:Ligation of the AND gate reporter(1.3kb) into pUC19 plasmid (2.6kb ) [F+pUC]. 3.9kb ligated band is visible.
**iii)LldP-R**:Ligation of LldP (1.7kb ) with LldR (0.9kb)=2.6kb. This band was not visible. **iv)Kill Switch unit**: Ligation of Holin-Endolysin (1.4kb) with Antiholin-Lamda Repressor (1.4kb) = 2.8kb. The ligated band is visible.*

#### Resuts

Ligation of TRAIL-Smac Fusion Protein+pUC, AND gate reporter +pUC and Kill Switch became successful. Ligation of LldP-R was not successful.

![](https://static.igem.org/mediawiki/2021/c/c3/T--IISER_Bhopal--Engineering_Success--Wet_Lab_Results--image_8.jpg)
![](https://static.igem.org/mediawiki/2021/3/3f/T--IISER_Bhopal--Engineering_Success--Wet_Lab_Results--image_9.jpg)

*Fig. 7:i)LEFT:Re-ligation of LldP-LldR and Kill switch. We got the desired band (2.6kb for LldP-R and 2.8kb for Kill Switch). ii)RIGHT: Ligation of Kill Switch (2.8kb) into pMB1 (3kb) through Sal1 and Xho1 sites. We got the ligated Kill Switch +pMB1 (5.8kb band ) in well 1 & 2.*

#### 4) Transformation of TRAIL-Smac +pUC & AND Gate+pUC -Blue-White screening

![](https://static.igem.org/mediawiki/2021/1/17/T--IISER_Bhopal--Engineering_Success--Wet_Lab_Results--image_10.jpg)
![](https://static.igem.org/mediawiki/2021/7/72/T--IISER_Bhopal--Engineering_Success--Wet_Lab_Results--image_11.jpg)

*Fig. 8:Blue White screening- i)DH5-α transformed with AND Gate Reporter (F+pUC ) & TRAIL-Smac FP (D+pUC) were plated in 100µg/mL Ampicillin plates with X-Gal & IPTG. Few White colonies were observed in each plate, along with blue colonies.
ii)No plasmid Control (white colonies) and pUC19 without insert Control (blue colonies).*

![](https://static.igem.org/mediawiki/2021/3/34/T--IISER_Bhopal--Engineering_Success--Wet_Lab_Results--image_12.jpg)
![](https://static.igem.org/mediawiki/2021/b/bb/T--IISER_Bhopal--Engineering_Success--Wet_Lab_Results--image_13.jpg)

*Fig. 9:After picking white colonies, the bacteria were re-plated on Ampicillin Plates with IPTG & X-Gal i)AND-Gate Reporter (F+pUC )- 100µg/mL Ampicillin
ii)TRAIL-Smac Fusion Protein (D+pUC).*

### Characterization of Kill Switch

Our Kill Switch is controlled by Lactate. It will kill bacteria under low lactate conditions and allow growth only if lactate concentration is more than 10 mM. As most solid tumors have a lactate concentration of approximately 10- 40mM, our kill switch will enable bacteria to live in the tumor microenvironment while not allowing them to live in normal tissues (lactate 1.3 to 3 mM). We successfully ligated the Kill Switch Components (Holin-Endolysin & Antiholin -Lamba Repressor) into pMB1 Plasmid.

![](https://static.igem.org/mediawiki/2021/1/12/T--IISER_Bhopal--Engineering_Success--Wet_Lab_Results--image_14.jpg)
![](https://static.igem.org/mediawiki/2021/a/a1/T--IISER_Bhopal--Engineering_Success--Wet_Lab_Results--image_15.jpg)

![](https://static.igem.org/mediawiki/2021/f/f2/T--IISER_Bhopal--Engineering_Success--Wet_Lab_Results--image_16.jpg)
![](https://static.igem.org/mediawiki/2021/3/36/T--IISER_Bhopal--Engineering_Success--Wet_Lab_Results--image_17.jpg)

*Fig. 10: Transformation of E. coli DH5-𝛂 with the Kill Switch (Holin-Antiholin-pMB1) and Plating in :(A)40mM lactate, 50µg/mL Spectinomycin (B)0mM lactate , 50µg/mL Spectinomycin (C)Control -No plasmid (No antibiotic) (D)Control - pMB1 (50µg/mL Spectinomycin)*

We could see a drastic difference in the growth of DH5-𝛂at 0mM Lactate and 40mM lactate plates. In 0mM lactate plates, the kill switch was operational, Holin and Endolysin were produced, killing the bacterium and preventing growth. Only one colony was observed in the 0mM lactate plate (could be an experimental error/ contamination). At 40mM concentrations of lactate, antiholin and lambda repressor were produced, which inhibited the holin and endolysin. Thus bacteria were able to survive and form many colonies.

![](https://static.igem.org/mediawiki/2021/3/38/T--IISER_Bhopal--Engineering_Success--Wet_Lab_Results--image_18.jpg)
![](https://static.igem.org/mediawiki/2021/c/c8/T--IISER_Bhopal--Engineering_Success--Wet_Lab_Results--image_19.jpg)

![](https://static.igem.org/mediawiki/2021/3/3e/T--IISER_Bhopal--Engineering_Success--Wet_Lab_Results--image_20.jpg)
![](https://static.igem.org/mediawiki/2021/4/41/T--IISER_Bhopal--Engineering_Success--Wet_Lab_Results--image_21.jpg)

*Fig. 11: The growth of DH5-α transformed with Kill Switch+pMB1 at :(A)3mM lactate , 50µg/mL Spectinomycin (B)5mM lactate, 50µg/mL Spectinomycin (C)10mM lactate , 50µg/mL Spectinomycin (D)20mM lactate, 50µg/mL Spectinomycin*

Small bacterial colony growth was observed in each plate, 3mM and 5mM lactate plates had fewer colonies, and 20 mM had a large number of colonies. We think there is a little bit of leakiness in the kill switch functioning. This leakiness could be the action of a lambda repressor, whose secretion is controlled by lactate. Lambda repressor will reduce Holin production by binding to the cL promoter. This allows us to deliver the bacteria via the intravenous route, as the lambda repressor will delay the action of the kill switch enough for bacteria to localize to the tumor region. But we could observe a considerable increase in the number of colonies at 20mM lactate. So our kill switch allows optimal bacterial growth from 10 to 40 mM lactate conditions, typically observed in solid tumors.

CFU per ml = (No. of colonies \* Total dilution factor)/Vol. of culture plated in mL

Dilution Factor = **2**
Vol. of culture plated in mL =**0.1ml**

| Sl.No | Lactate concentration in mM | Number of colonies / Quarter of the plate   (n) | Total Colonies  (t = n\*4) | CFU /mL ( t\*2 /0.1 ml) |
| --- | --- | --- | --- | --- |
| 1 | 0 | Nil | 1 (only 1 colony) | 20 |
| 2 | 3 | 4 | 16 | 3.2 \*102 |
| 3 | 5 | 7 | 28 | 5.6\*102 |
| 4 | 10 | 20 | 80 | 1.6 \*103 |
| 5 | 20 | 242 | 968 | 1.93\*104 |
| 6 | 40 | 285 | 1140 | 2.28\*104 |

**OD Measurement & Growth Curve of Bacteria with Kill Switch**

| Time/Lactate concentration | 0 mM Lactate | 1.5 mM Lactate | 5mM Lactate | 10mM Lactate | 20mM Lactate | 40mM Lactate |
| --- | --- | --- | --- | --- | --- | --- |
| 0.5 hr | 0.015 | 0.050 | 0.085 | 0.088 | 0.122 | 0.190 |
| 1 hr | 0.090 | 0.134 | 0.120 | 0.103 | 0.227 | 0.277 |
| 1.5 hr | 0.104 | 0.164 | 0.227 | 0.196 | 0.344 | 0.384 |
| 2 hr | 0.158 | 0.230 | 0.330 | 0.297 | 0.447 | 0.513 |
| 2.5 hr | 0.163 | 0.260 | 0.350 | 0.389 | 0.512 | 0.590 |
| 3 hr | 0.175 | 0.315 | 0.393 | 0.440 | 0.593 | 0.660 |
| 3.5 hr | 0.226 | 0.386 | 0.412 | 0.495 | 0.659 | 0.875 |
| 4 hr | 0.254 | 0.380 | 0.431 | 0.529 | 0.743 | 0.912 |
| 4.5 hr | 0.286 | 0.434 | 0.481 | 0.610 | 0.805 | 0.960 |
| 5 hr | 0.312 | 0.386 | 0.486 | 0.734 | 0.868 | 0.992 |
| 5.5 hr | 0.330 | 0.431 | 0.586 | 0.844 | 0.905 | 1.066 |
| 6 hr | 0.354 | 0.423 | 0.598 | 0.945 | 1.008 | 1.158 |

*Table:1)OD measurement after inoculation 100 µL primary culture into 5mL fresh LB with 50µg/mL Spectinomycin and different lactate concentrations (0mM,1.5mM,5mM,10mM,20mM,40mM)*

![](https://static.igem.org/mediawiki/2021/9/99/T--IISER_Bhopal--Engineering_Success--Wet_Lab_Results--image_22.jpg)

*Fig. 12:Growth Curve of bacteria (DH5-α) with kill switch at different lactate concentrations.*

## Drylab Results

To get a holistic view of our project, we did a combination of mathematical, structural and simulation modelling. Here we compile our results from the various models and the questions that motivated us to build them.

### Smac Internalization Kinetics

The rate of internalisation of Smac(n7)-R8 in the cancer cells plays an important role in inducing apoptosis. We used COPASI to build a mathematical model using Michaelis-Menten kinetics.

### Molecular Dynamics Simulation of Smac Internalization

Due to delayed access to the wet lab, we wanted to check the internalization of the Smac-CPP (Cell-Penetrating Peptide) construct through the lipid bilayer. For this, we decided to run molecular dynamics simulations using GROMACS and CHARMM force fields on a high performance computing cluster.

### Caspase Cascade Kinetics

This is a mathematical model made using COPASI that deals with the TRAIL-apoptosis machinery after fusion TRAIL binds with the death receptors present in the plasma membrane. This model shows an increase in the concentration of activated caspase3 after activation of death receptors.

### Kill Switch Time Delay

A major question that we came across while designing our kill switch is the time it takes to get activated in the absence of lactate. For this, we mathematically modelled our system using COPASI and found that the system has a time delay of 20 minutes (considered as an underestimation).

### Structural Modelling of TRAIL

Since our fusion construct is a polyprotein that gets cleaved to form Smac and TRAIL, we were motivated to check whether the linker residues impact the structure and thereby the function of TRAIL after cleavage. The structures of the native and fusion constructs were quite similar, hence we may conclude that there is a high chance that the active and functionally relevant domains in our fusion construct TRAIL will remain the same as that of native TRAIL.

### The more about the same can be found on our [Modelling page.](https://2021.igem.org/Team%3AIISER_Bhopal/Model)