iGEM 2022 — Team rec-chennai

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## March

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar21.png)

Introduction to iGEM

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar23.png)

Team recruitment began

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar25.png)

Interviews were conducted for students who showed interest in joining iGEM.

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar30.png)

The whole iGEM team was finalised.

## April

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar1.png)

First official offline meet-up was held.

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar4.png)

Lectures from faculties on iGEM protocols

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar6.png)

1st brainstorming session

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar8.png)

Literature review was done.

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar9.png)

Had a 1st online meet-up with the seniors for getting to know about iGEM and their experience.

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar12.png)

Opened the iGEM lab and had the stock check done.

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar15.png)

Brainstorming session.

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar16.png)

Literature review was done

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar20.png)

Official group picture was taken.

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar22.png)

Fumigation of lab.

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar25.png)

1st lab discussion.

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar28.png)

Literature review was done.

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar30.png)

Stock check

## May

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar3.png)

Brainstorming session

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar6.png)

Troubleshoots

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar10.png)

Literature review

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar12.png)

Advice from investigators

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar14.png)

Discussion with instructors

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar18.png)

Stock check

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar20.png)

Safety Check

## June

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar9.png)

Registration of the students into the iGEM REC Chennai Team.

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar11.png)

Accepted as member of REC-CHENNAI iGEM team.

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar18.png)

Arrival of *E.coli* Alpha strain.

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar19.png)

Grant submission

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar23.png)

Subculture.
 Safety forms submitted.

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar24.png)

iGEM kit received

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar25.png)

iGEM kit unboxed

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar28.png)

Subculture.

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar29.png)

Submitted safety and security grant.

## July

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar1.png)

Subculture, online meet for team discussion.

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar5.png)

Subculture and testing viability of mother culture and discussion about the biochemical assay.

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar8.png)

Online meet.

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar9.png)

Subculture and fumigation of lab.

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar12.png)

Offline meetup for work discussion.

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar13.png)

Subculture

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar14.png)

DH5 alpha test and reactor building discussions.

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar16.png)

Subculture and confirmation tests

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar21.png)

Online meet discussion for AIIMS.

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar24.png)

AIIMS presentation

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar26.png)

Collecting K12 and DH5 alpha strain from IITM.

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar27.png)

Madras University meet with Dr. B. Anandan

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar29.png)

SBOA School and Junior College HP, Online meet with Team Waterloo.

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar30.png)

Subculture and stock check

## August

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar3.png)

Online meet to discuss for sponsorship possiblities & college level event

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar4.png)

Meet with our advisor Mr. Harikumar, discussion with Dr. P. Saravanan. Online meet with Madlyn Thompson.

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar5.png)

Night stay for lab work: Biofilm assay and Subculture

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar6.png)

Sacred Heart School HP

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar8.png)

Rehersal for game launch program.

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar9.png)

CURCOLI game launch and subculture.

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar12.png)

Sponsorship meet with Textan MD.

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar14.png)

Online meet with Mr. Vijay Bhoopalan.

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar17.png)

Bhavan's Rajaji Vidyashram School HP

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar20.png)

Subculture and Biofilm new protocol attempt

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar22.png)

Successfully biofilms formed

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar23.png)

Discussion for beach HP and subculture

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar25.png)

Kalpakkam Scientist meet with Dr. Venkat Nancharaiah and Dr. Kiran

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar27.png)

Protocol Optimization: MTT assay, pNPP assay and subculture

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar28.png)

Edward Elliots Beach HP

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar29.png)

Meet with iGEM-Munich

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar30.png)

Protocol optimization: Growth curve, Cd toxicity, subculture and stock check

## September

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar2.png)

Promotion video uploaded

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar6.png)

Meet with Twist Bioscience

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar7.png)

Placed order in Twist Bioscience

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar9.png)

Night stay for Protocol Optimization: Transformation, HRP assay and subculture

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar10.png)

Primers synthesis order

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar15.png)

Final Safety Form submitted

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar18.png)

Online meet with Advisors

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar19.png)

Meet with IIT Delhi and subculture

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar20.png)

Received primers

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar21.png)

Night stay for Protocol Optimization: Transformation, Protein Quantitative assays.

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar24.png)

Meet with Mr. Deepak Kumar

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar26.png)

Received parts from Twist bioscience and subculture

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar28.png)

Lab Activity: PCR and analysis of plasmid.

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar29.png)

Lab Activity: Restriction Digestion, Ligation and Plasmid mini prep.

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar30.png)

Lab Activity: Transformation.

## October

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar1.png)

Lab Activity: Isolation of Colonies and subculture.

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar3.png)

Reactor setup

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar4.png)

Biofilm assay

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar5.png)

Characterization: CV, Congo red, Saffranin

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar6.png)

Characterization: Growth Curve, Cd toxicity assay and subculture

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar7.png)

Characterization: Samples given for AAS assay and SEM, MTT assay

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar8.png)

Characterization: MTT assay, pNPP assay

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar10.png)

Characterization: HRP assay

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar11.png)

Final wiki edition

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Final wiki edition, Wiki, Registry, Judging form freeze

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar13.png)

Team meet

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar14.png)

Poster and PPT Making

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar17.png)

Poster and PPT Making

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar19.png)

Presentation preparation

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar22.png)

Team meet

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar26.png)

Grand Jamboree

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar27.png)

Grand Jamboree

![](https://static.igem.wiki/teams/4509/wiki/notebook/calendar28.png)

Grand Jamboree

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{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
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[Overview](#overview)
[Module 1 : Parts Engineering](#module1)
[Iteration 1](#iteration11)
[• Design](#design11)
[• Build](#build11)
[• Test](#test11)
[• Learn](#learn11)
[Iteration 2](#iteration12)
[• Design](#design12)
[• Build](#build12)
[• Test](#test12)
[• Learn](#learn12)
[Module 2 : Reactor Engineering](#module2)
[Iteration 1](#iteration21)
[• Design](#design21)
[• Build](#build21)
[• Test](#test21)
[• Learn](#learn21)
[Iteration 2](#iteration22)
[• Design](#design22)
[• Build](#build22)
[• Test](#test22)
[• Learn](#learn22)

## Overview

---

The Engineering Cycle is a step-by-step method for developing and maintaining a biological system. It is used to identify problems and narrow down the concept to a single efficient prototype. The cycle begins with design, in which the team brainstorms an idea and draughts a model design. The next step is to build the prototype and bring the design to life. The third step is to put the prototype through its paces and check for errors and efficiency. The third step is followed by the learning cycle, which corrects any errors or inefficiencies. The cycle is repeated until the project is optimized with the final test run.

## Module 1 : Parts Engineering

---

### Iteration 1

##### Design

Our goal is to facilitate bioprecipitation of cadmium ions from industrial effluent for which the engineered bacteria should satisfy two major conditions. The bacteria should be able to form biofilm and it should express acid phosphatase enzyme. From the organisms on the white list cataloged by iGEM, we chose *E.coli* DH5 alpha and *E.coli* K12 MG1655 strains to check if it would fulfill the conditions[1]. Finally after literature mining and observing biofilm formation in both the strains, *E.coli* K12 strain was selected. The structure and other physiochemical properties of acid phosphatase enzyme were studied in detail[2]. Dephosphorylation of the substrate takes place when acid phosphatase binds with the phosphate substrate. Bioprecipitation takes place as the metal phosphate is not soluble in aqueous solution. We were introduced to curli biogenesis by Dr.Saravanan during the internal iHP meet. Then, we found out that the ability of the E. coli to produce biofilm is majorly dependent on csgA[3][4].Our initial plan was to modify BBa\_K1404006 part submitted by the team INSA Lyon 2010 by linking acid phosphatase gene to csgA with the help of a rigid linker[5]. Thus, the fusion protein will be present attached to the curli of biofilm and will not get washed away by effluents.

##### Build

We chose the Biobrick RFC[10] standard as the majority of parts in the Registry database are RFC[10] compatible. Scar sequence for Biobrick RFC[10] was identified and used during virtual plasmid construction with the parts in Benchling. We had the opportunity to meet Dr.Anandan sir during this phase. He pointed out the fact that it would be difficult for the bacteria to achieve a curli- mediated transport of the phosphatase enzyme since the fusion protein shouldn’t affect the biofilm strength and he asked us to predict the fusion protein structure and check the binding strength of the fusion protein.

##### Test

To predict the structures and binding strength, we used I-TASSER .We computationally modelled the fusion protein (csgA aphA). Then,We received five predicted models from I-TASSER with a c-score in the range -4.7 to -3.2.

##### Learn

As suggested by Dr.Anandan during our iHP meeting, the result should be in such a way that the expression of the enzyme should not affect the biofilm production. We were working on *E.coli* K12 strain which is a poor biofilm forming strain. So the parts that we use in our project should enhance biofilm formation and express the enzyme at the cell surface.

### Iteration 2

##### Design

From what we learnt, it is clear that the parts we choose must enhance biofilm formation and express acid phosphatase enzyme at the cell surface in the desired *E.coli* K12 MG1655 strain. Hence we planned to modify the composite part BBa\_K2229300 submitted by the team iGEM17\_TAS\_Taipei by replacing the BBa\_J23100 promoter with another strong promoter to reduce the burden on the organism and OmpR upregulation will facilitate csgD overexpression which in turn aids in curli biogenesis. This curli composite part BBa\_K4509469 is joined with another composite part BBa\_K4509669 consisting of a cadmium metal sensing promoter (BBa\_K896008), strong RBS (BBa\_B0034), cell surface tag (BBa\_K103006) linked to acid phosphatase gene (BBa\_K4509369), with a double terminator (BBa\_B0015). This composite part has been designed to express the acid phosphatase enzyme on the cell surface.

![](https://static.igem.wiki/teams/4509/wiki/engineering/eng1.jpeg)

![](https://static.igem.wiki/teams/4509/wiki/engineering/eng2.jpeg)

![](https://static.igem.wiki/teams/4509/wiki/engineering/eng3.jpeg)

##### Build

According to the design, the parts were constructed using Biobrick RFC[10] standard. We chose the RFC[10] standard because the curli composite part that we chose to modify followed the RFC[10] standard and the parts in the cell surface aphA composite part were all RFC[10] compatible. The scar sequence for RFC[10] was used. We were able to virtually construct the plasmid with the help of Benchling. We are being mentored by iGEM team member Deepak Kumar, who gave us insights about the standard and assembly methods and helped us in construction. OmpR upregulation facilitates csgD overexpression which in turn helps in curli biogenesis and the ompA-acid phosphatase fusion protein is expressed on the cell surface.

##### Test

To predict the structures and binding strength, we used I-TASSER .We computationally modelled the fusion protein (OmpA aphA). Then, We received five predicted models from I-TASSER with a c-score in the range -4.2 to -3.4.

##### Learn

As expected, the overexpression of csgD enhanced biofilm formation. From the information obtained by computationally modeling (I TASSER) the fusion protein, the c-scores of the fusion protein after modification have better scores when compared.

## Module 2 : Reactor Engineering

---

### Iteration 1

##### Design

![](https://static.igem.wiki/teams/4509/wiki/figure-5-primary-lab-adaptable-reactor-setup.jpg)

The primary goal of project CURLIM is to create a method for removing cadmium from effluent. We intend to construct a bioreactor that facilitates the growth of genetically modified E. coli K12 to form biofilm, which will produce acid phosphatase. After that, the enzyme will dephosphorylate the phosphate monomers (substrates) to produce free phosphate. Cadmium phosphate will precipitate once the free phosphate bonds to the cadmium ions in the effluent. We are designing
a laboratory Adaptable Moving Bed Biofilm Reactor in order to identify the key requirements for our user-level bioreactor and make design predictions (MBBR).

Dr. Venkat of the Madras Atomic Power Station in Kalpakkam gave the Team a unique perspective on Lab Scale Reactor Construction when they spoke with him. Dr. Anandan, Assistant Professor at Madras University's Tharamani Campus, also gave us constructive feedback on the environmental and reactor conditions to take into account, as well as the Enzyme Kinetics parameters. As a result, we realized that the Field Adaptable Moving Bed Biofilm Reactor (MBBR) needed to be as simple and configurable as possible so that end-users could build it with whatever resources they had available.

##### Build

Our design includes a cylindrical tank that will serve as the Primary Lab Adaptable Reactor Setup's primary culturing tank. The GAC OR plastic bio carriers will serve as a surface for the growth of biofilms. The first step was to drain the media solution after the main tank had produced the required quantity of biofilm, allowing the bio carriers to settle. The feed, which contains synthetic effluent, phosphate substrate, and microbe nutrients, is then added to the tank with the bio carriers. To maintain a consistent contaminant composition, synthetic effluent was created in the laboratory and used throughout the investigation. The main apparatus, which was kept, consisted of an aerator with membrane, a 4L cylinder, a 2-way peristaltic pump to control inlet and output flow, and a half L conical flask.

##### Test

There was an evidential growth of biofilm but it was inefficient. The secreted acid phosphatase enzyme interacted with the heavy metals in the feed, resulting in the formation of Cd-Phosphate complexes and sediment. Thus, bio-precipitation was performed in the MBBR.

##### Learn

The efficient formation of biofilms necessitates an optimum temperature of 37 degrees Celsius, which the primary adaptable reactor setup could not provide.

### Iteration 2

##### Design

![](https://static.igem.wiki/teams/4509/wiki/engineering/reactor-design.jpeg)

The team conducted additional research to optimize the environmental and reactor conditions. These investigations resulted in an improved version of the Laboratory Adaptable Moving Bed Biofilm Reactor (MBBR) which is divided into two parts. The enhanced setup incorporated a water bath thermal insulation layer outside the reactor tank to keep the temperature between 28 and 37 degrees Celsius, and another was an inner zone of the reaction site. Industries may effectively choose the best design that suits their demands by offering essential growing conditions and design recommendations. More information can be found on our [Bioreactor](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27bioreactor%27%29%20%7D%7D) page!

##### Build

A 4 L lab-scale batch reactor was constructed using commercially available Plexi glass vessels. It was split into two sections. One was an outside water bath thermal insulation layer to keep the temperature between 28 and 37 degrees Celsius, and the other was an inner zone of the
reaction site. The air grid and influent pipes were installed at the reactor's bottom, as shown in Figure. Air was supplied from the reactor's bottom at a calculated flow rate of 1.8 m3/h to provide oxygen for the microbial mass's biological activity and to fluidize the carriers. Two separate peristaltic pumps are used to feed the influent to both reactors while maintaining their respective HRTs. To separate solids from effluent, two half-liter sludge settlers are built downstream of the reactors.

The biofilm carriers used were made of GAC (0.4-0.5 g/cc) or PE plastic (density 0.94-0.96 g/cm3) and were designed to provide protected surface areas for microbial biofilm development. The influent source was carcinogenic Cadmium-containing synthetic wastewater.

##### Test

There was significant growth of biofilm. The secreted acid phosphatase enzyme interacted with the heavy metals in the feed, resulting in the formation of Cd-Phosphate complexes and sediment. Thus, bio-precipitation was executed in the MBBR.

##### Learn

Laboratory Adaptable Moving Bed Biofilm Reactor (MBBR) that would simulate the conditions that industries would use to treat cadmium ions considering that this setup would later guide us in determining the necessary specifications for our user-level bioreactor as well as making design predictions.

### References ---

1. Brombacher, E., Baratto, A., Dorel, C., & Landini, P. (2006). Gene Expression Regulation by the Curli Activator CsgD Protein: Modulation of Cellulose Biosynthesis and Control of Negative Determinants for Microbial Adhesion. Journal of Bacteriology, 188(6), 2027-2037. <http://doi.org/10.1128/JB.188.6.2027-2037.2006>
2. Evans, M. L., & Chapman, M. R. (2014). Curli biogenesis: order out of disorder. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, 1843(8), 1551-1558.
3. <https://www.uniprot.org/uniprotkb/P0AE22/entry>
4. [http://2014.igem.org/Team:INSA-Lyon/Biology](http://2014.igem.org/Team%3AINSA-Lyon/Biology)
5. [http://2017.igem.org/Team:TAS\_Taipei](http://2017.igem.org/Team%3ATAS_Taipei)
6. Martinez-Hackert, E., & Stock, A. M. (1997). The DNA-binding domain of OmpR: crystal structures of a winged helix transcription factor. Chemistry & Biology, 4(2), 162. doi:10.1016/s1074-5521(97)90269-6
7. Olsén, A., Wick, M. J., Mörgelin, M., & Björck, L. (1998). Curli, fibrous surface proteins of Escherichia coli, interact with major histocompatibility complex class I molecules. Infection and immunity, 66(3), 944-949.
8. Reinke, A A, and J E Gestwicki. “Insight into amyloid structure using chemical probes.” Chemical biology & drug design., U.S. National Library of Medicine, June 2011, <www.ncbi.nlm.nih.gov/pubmed/21457473>.
9. Reisner, A., Haagensen, J. A., Schembri, M. A., Zechner, E. L., & Molin, S. (2003). Development and maturation of Escherichia coli K-12 biofilms. Molecular microbiology, 48(4), 933-946.
10. J. Xing, R. Hickey, Response in performance of the GAC-fluidized bed reactor process for BTX removal to perturbation in oxygen and nutrient supply, International Biodeterioration & Biodegradation, Volume 33, Issue 1,1994, Pages 23-39, ISSN 0964-8305, [https://doi.org/10.1016/0964-8305(94)90053-1](https://doi.org/10.1016/0964-8305%2894%2990053-1).
11. Magdum, Sandip & V., Kalyanraman. (2019). Evaluation of High Rate MBBR to Predict Optimal Design Parameters for Higher Carbon and Subsequent Ammoniacal Nitrogen Removal. Current science, 116. 2083-2088. 10.18520/cs/v116/i12/2083-2088.
12. Brackin, M.J., McKenzie, D.E., Hughes, B.M. et al. Laboratory-scale evaluation of fluidized bed reactor technology for biotreatment of maleic anhydride process wastewater. Journal of Industrial Microbiology 16, 216-223 (1996). <https://doi.org/10.1007/BF01570024>.
13. Yu N, Zhang D, Lei Y, Wang J, Dong Y, Chen Y. Innovative Coating-Etching Method of Biocarrier Fabrication for Treating Wastewater with a Low C/N Ratio. Polymers, 2022 Jul 25;14(15):3010.

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{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
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## Experiments

* [1](#MediaPrep "MEDIA PREPARATION")
* [2](#Subculture "SUBCULTURE")
* [3](#MiniPrep "MINI PREP")
* [4](#3aAssembly "3A Assembly")
* [5](#PCR "PCR")
* [6](#AgaroseGelElectrophoresis "Agarose Gel Electrophoresis")
* [7](#RestrictionDigestion "Restriction digestion")
* [8](#Ligation "Ligation")
* [9](#CompetentCellPreparation "Competent Cell Preparation")
* [10](#BiuretTest "Biuret Test")
* [11](#pNPP "pNPP Phosphatase Assay")
* [12](#BiofilmFormation "BIOFILM FORMATION ON DIFFERENT SURFACES")
* [13](#CongoRedAssay "Congo Red Assay")
* [14](#BiofilmAssay "Biofilm Assay Using Safranin")
* [15](#HRPAssay "HRP Assay")
* [16](#BiofilmCVassay "Biofilm Assay Using Crystal Violet")
* [17](#CdToxicityTest "Cadmium Toxicity Test")
* [18](#MTTAssay "MTT Assay")
* [19](#SterilizationMethods "Sterilization Methods")

### MEDIA PREPARATION ---

##### Materials Required :

1. LB broth mix
2. LB Agar
3. Distilled water
4. Plates and conical flasks

##### Procedure :

1. Mix 25g of LB broth powder in 1L of Distilled water.
2. Mix 40 g of LB agar in 1L of distilled water.
3. Autoclave and cool.
4. Pour the broth into a conical flask.
5. Pour the agar mixture onto a plate and let it set.

### SUBCULTURE ---

#### INOCULATION

##### Materials Required :

1. Autoclaved LB broth
2. Pipette tips

##### Procedure :

1. Pipette out bacterial cells and inoculate into LB broth.
2. Close the conical flask with non absorbent cotton.
3. Incubate the flask in an incubator at 37oC.

#### SPREAD PLATE

##### Materials Required :

1. Dry agar plate (with/without antibiotics)
2. Sterile spreader
3. Parafilm tape
4. Transformed cells in LB broth grown for 1 hour at 37oC in the shaker.

##### Procedure :

1. Pipette out of transformed cells in LB Broth onto the center of the surface of an agar plate.
2. Use a sterile plastic spreader.
3. Spread the sample evenly over the surface of agar using the spreader.
4. Wrap the Petri dish with parafilm.
5. Incubate the plate at 37°C for 8-12 hours.

### MINI PREP ---

##### Materials Required :

1. P1 - Resuspension buffer- 50mM Tris•HCl (pH = 8), 10mM EDTA, 100μg/ml RNase A
2. P2 - Lysis buffer - 200mM NaOH, 1% SDS
3. P3 - Precipitation buffer - 3M potassium acetate (pH = 5.5)

##### Procedure :

1. Grow 2 to 6ml culture O/N
2. Transfer 2ml (at a time) to 2ml microcentrifuge tube(s) and pellet at 14000 rpm for 5 minutes. Discard supernatant and repeat as necessary
3. Resuspend in 300μl ice-cold P1
4. Add 300μl P2, gently invert 3x. Quickly add 300μl P3 and invert 3x
5. Spin at 14000 rpm for 10 minutes at room temperature
6. Retain supernatant in 1.5ml microcentrifuge tube
7. Add 650μl of 100% isopropanol, gently invert, and incubate for 10 minutes at room temperature
8. Spin at 14000 rpm for 10 minutes, then discard supernatant
9. Wash pellet with 500μl of cold 70% ethanol
10. Spin at 14000 rpm for 5 minutes
11. Discard the supernatant. Carefully tap the tube to remove remaining ethanol. Dry pellet in vacuum.
12. Resuspend the pellet in 20μl of sterile ddH2O or TE buffer. Nanodrop to determine concentration. Store at -20°C

### 3A Assembly ---

##### Materials Required :

1. Two Parts to be joined A and B: Miniprepped
2. Linearized Plasmid Backbone
3. Restriction enzyme: EcoRI, XbaI, SpeI, PstI, DpnI
4. Buffer (10X buffer-50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM DTT, pH 7.9 at 25°C.)
5. BSA
6. NFW

##### Procedure :

1. Enzyme Master Mix for Plasmid Backbone
  a. 5 ul Buffer
  b. 0.5 ul BSA
  c. 0.5 ul EcoRI-HF
  d. 0.5 ul PstI
  e. 0.5 ul DpnI
  f. 18 ul NFW
2. Enzyme Master Mix for Part A
  a. 5 ul Buffer
  b. 0.5 ul BSA
  c. 0.5 ul EcoRI-HF
  d. 0.5 ul SpeI
  e. 18.5 ul NFW
3. Enzyme Master Mix for Part B
  a. 5 ul Buffer
  b. 0.5 ul BSA
  c. 0.5 ul XbaI
  d. 0.5 ul PstI
  e. 18.5 ul NFW
4. Digest Plasmid Backbone by adding 4 ul linearized plasmid backbone (25ng/ul for 100ng total) and 4 ul of Enzyme Master Mix for plasmid at 37oC for 30 mins.
5. Digest Part A by adding 4 ul Part A (25ng/ul for 100ng total) and 4 ul of Enzyme Master Mix for part A at 37oC for 30 mins.
6. Digest Part B by adding 4 ul Part B (25ng/ul for 100ng total) and 4 ul of Enzyme Master Mix for part B at 37oC.
7. Add 2ul of digested Plasmid Backbone (25 ng)
8. Add equimolar amount of Part A (EcoRI-HF SpeI digested) fragment (< 3 ul)
9. Add equimolar amount of Part B (XbaI PstI digested fragment) (< 3 ul)
10. Add 1 ul T4 DNA ligase buffer. Note: Do not use quick ligase
11. Add 0.5 ul T4 DNA ligase
12. Add NFW upto 10 ul
13. Ligate at 16o C for 30 mins.
14. Transform with 1-2 ul of product

### PCR ---

##### Procedure :

##### 1. PCR Setup

2X master mix
Forward primer (10 picomol/Ul)
Reverse primer (10 picomol/Ul)
cDNA (100ng)
NFW

10 ul
1 ul
1 ul
2 ul
6 ul
20 ul

##### 2. Temperature Profile

Initial Denaturation
Denaturation
Annealing
Extensive
Final temperature

: 95oC - 5 mins
: 95oC - 30 sec
: 20 sec (Ta = Tm - 5)
: 72oC - 1kb 1 min (depends on length)
: 72oC - 5 to 10 min

### Agarose Gel Electrophoresis ---

##### Materials Required :

1. TAE (EDTA, Acetate, Tris-base)
2. Agarose
3. Ethidium bromide
4. Casting tray
5. Well combs
6. Voltage source
7. Gel box
8. UV light source
9. Microwave

##### Procedure :

1. Make 1% Agarose Gel. (Mix 1g of Agarose powder with 100mL of 1X TAE buffer)
2. Microwave for 1-3 mins until the agarose is completely dissolved.
3. Let the agarose solution cool down.
4. Add ethidium bromide (EtBr) to the agarose solution of approximately 0.2-0.5 μg/mL (usually about 2-3 μl of lab stock solution per 100 mL gel). EtBr binds to the DNA and allows you to visualize the DNA under ultraviolet (UV) light.
5. Pour the agarose into a gel tray with the well comb in place.
6. Let the agarose solution set.
7. Once solidified, place the agarose gel into the gel box (electrophoresis unit).
8. Fill the gel box with 1xTAE (or TBE) until the gel is covered.
9. Add loading buffer to each of your DNA samples.(Loading buffer serves two purposes: 1) it provides a visible dye that helps with gel loading and allows you to gauge how far the DNA has migrated; 2) it contains a high percentage of glycerol that increases the density of your DNA sample).
10. Carefully load a molecular weight ladder into the first lane of the gel.
11. Carefully load your samples into the additional wells of the gel.
12. Run the gel at 80-150 V until the dye line is approximately 75-80% of the way down the gel.Turn OFF power, disconnect the electrodes from the power source, and then carefully remove the gel from the gel box.
13. Using any device that has UV light, visualize your DNA fragments.
14. Using the DNA ladder in the first lane as a guide , you can infer the size of the DNA in your sample lanes.

#### Gel Extraction

##### Procedure :

1. Once you have run your gel, move it to an open UV box, remove it from any gel tray as plastic will block much of the UV and with a clean, sterile razor blade, slice the desired DNA fragment from the gel.
2. Place the gel in a labeled microfuge tube.
3. Using a scale, weigh the gel fragment because it is directly proportional to its liquid volume and this is used to determine how much of each buffer to add during the DNA isolation step.
4. Add 200 μL Binding Buffer (NTI) per 100 mg gel.
5. Incubate the mixture at 50ºC for 5-10 minutes or until the gel has completely melted.
6. Add the above mix to a EZ column and let it sit for 2 mins, and centrifuge at 11,000 x g for 30s at room temperature.
7. Discard liquid and add 750 μL of wash buffer and centrifuge at 10000 rpm for 2min.
8. Repeat the step twice and discard the flow-through liquid and re-use the collection tube.
9. Place the EZ column into a clean 1.5 mL eppendorf tube. Add 15-30 μL of Elution Buffer (10 mM Tris-HCl, pH 8.5) directly onto the column matrix and incubate at room temperature for 1 minute. Centrifuge for 1 minute at 11,000 x g to elute DNA.

### Restriction digestion ---

##### Procedure :

1. Setup

Template
Restriction enzyme 1
Restriction enzyme 2
5X buffer
NFW

10 ul
1 ul
1 ul
5 ul
8 ul
25 ul

2. Incubate at 37o C for 2hours.
3. Run Gel electrophoresis

### Ligation ---

##### Procedure :

1. Setup

Vector
Insert
T4 DNA ligase
2X buffer
NFW

1 ul
1 ul
1 ul
5 ul
2 ul
10 ul

2. The ligated product is in 10 ul aliquot.

### Competent Cell Preparation ---

##### Materials Required :

1. LB miller broth
2. E coli cells
3. Conical flasks
4. Falcon tubes
5. Ice and ice box
6. CaCl2 solution (20mM and 0.1M)
7. 80mM MgCl2 solution
8. 1.5mL microcentrifuge tubes

##### Procedure :

##### Chemical Method :

1. Take 10 mL LB broth and inoculate it with E. coli and let it grow for 12 hours. Check OD600.
2. Transfer the colony in 50mL LB miller broth in 250mL (1:5 ratio) conical flask.
3. Now take OD after 30-60 mins until it reaches 0.35-0.4.
4. Transfer culture to sterile ice-cold falcon tubes. Cool culture to 0-4oC by incubating it on ice for 10 minutes.
5. Centrifuge the culture at 4o C at 4100rpm for 10 minutes.
6. Decant the medium and resuspend the cell pellet in 30mL of ice-cold MgCl2 and CaCl2 solution (80mM MgCl2 and 20mM CaCl2) by gentle swirling.
7. Again centrifuge at 4100rpm for 10 minutes at 4oC.
8. Decant the media and resuspend in 2mL of ice-cold 0.1M CaCl2 for each culture.
9. Make aliquots of 100 ul each and store at -80oC.

##### Transformation

##### Materials Required :

1. Competent E coli cells
2. Ligated products
3. 1.5 mL microcentrifuge tubes
4. Ice and icebox
5. LB miller broth
6. LB agar with appropriate antibiotic plate (Chloramphenicol)
7. Micropipette and tips

##### Procedure :

1. Mix 10 ul of ligated products with 100 ul aliquots of competent cells in 1.5mL centrifuge tubes.
2. Leave on ice for 15-30 minutes.
3. Heat shock the mixture in a water bath for 45 seconds at 42o C.
4. Keep in ice for 5 minutes.
5. Add 1 ul of LB broth (without antibiotics) and let it grow in the shaking incubator for 45-60 minutes.
6. Spread 50 ul on the Chloramphenicol antibiotic plate and place it in a 37o C static incubator.
7. For the remaining solution, centrifuge at 4000 rpm for 4 minutes and resuspend in 50 ul LB broth in case you want to spread all transform cells or either store the remaining solution at -80o C.
8. Usually, colonies appear after 12-13 hours.

### BIURET TEST ---

##### Materials Required :

1. 1% alanine and 5% albumin or egg white (as positive control)
2. Biuret reagents(Copper sulfate (CuSO4), sodium hydroxide (NaOH) and sodium-potassium tartrate).
3. Deionised water (as negative control)
4. Dry test tubes
5. Water bath
6. Pipettes

##### Procedure :

1. Biuret reagent is prepared by dissolving copper sulfate and sodium,potassium tartrate in sodium hydroxide solution. To this solution potassium iodide is added and the volume is made up to 1L with sodium hydroxide.
2. Working standard - it is prepared by mixing protein (egg albumin) with distilled water.
3. To test tubes add 0.5 - 2.5 ml of working standard solution and make it up to 3 ml with distilled water.
4. Then 4 ml of biuret solution is added to all the test tubes.
5. The test tubes are mixed well and incubated at room temperature for 10 mins.
6. Similar procedure is followed for test solutions (unknown solution).
7. The purple colour development is measured at 520 nm.
8. Plot a graph between the protein concentration and optical density. This helps in identifying the protein concentration in the test solution (unknown concentration).

### pNPP Phosphatase Assay ---

To check the activity of the phosphatase enzyme.

##### Procedure

##### Materials Required :

1. Assay Buffer-neutral
2. Assay Buffer-acidic
3. Cell Lysis Buffer
4. Stop Buffer
5. Enzyme / Sample (1 Unit/mL) (Reconstitute enzyme with 1 mL of assay buffer)
6. pNpp Substrate (5 µl of substrate per 96-well)
7. Microplate reader
8. 96-well plates with clear flat-bottom
9. Dulbecco's Phosphate-Buffered Saline (PBS)

##### Sample Preparation :

1. Gently aspirate the cell culture medium.
2. Wash the cells twice with Dulbecco's Phosphate-Buffered Saline(PBS).
3. Lyse the cells with appropriate amount of cell lysis buffer (0.5 ml for 35 mm dish).
4. Centrifuge the cell lysate at 14,000rpm for 5 min at 4o C. Transfer the supernatant to a new tube.
5. Perform protein assay to determine total protein concentration in the lysate.
6. We recommend performing several dilutions in the assay buffer to ensure your readings fall within the standard range.

##### Assay procedure :

1. Apply 45 µl of each enzyme in duplicate to each of a 96-well plate.
2. Add 5 µl of substrate per well and incubate in the dark for 15-60 min.
3. Stop the reaction by adding 50 µl of stop buffer to each well. Mix and measure the absorbance on ELISA plate reader with a test wavelength at 405 nm and a reference wavelength at 630 nm. Subtract the 630 nm reference absorbance from the 405 nm measurement.

### BIOFILM FORMATION ON DIFFERENT SURFACES ---

##### Materials Required :

1. E.coli stock culture.
2. LB-MES broth.
3. 96 well plate.
4. Thermostable static incubator.
5. Orbital shaker.

##### On Plastic Biocarriers.

##### Procedure :

1. The initial E.coli stock culture was prepared by adding 2 ml inoculum into 20 ml standard LB-MES broth with a pH of 5.5 and allowing the E.coli to grow overnight (16-18 hrs) at 28oC under orbital shaker at 120 rpm.
2. Next day, the optical density (OD) at 600nm was checked. Value at 600nm must be 2.0 or dilute it.
3. The culture was diluted in the ratio of 1:100 with 20 ml of fresh LB - MES broth at a pH of 7.4 and 0.5 ml of culture in a conical flask.
4. Then add GAC (0.4-0.5 g/cc) or PE plastic (0.94-0.96 g/cm3) bio carriers into the conical flask.
5. After 48 hrs the biocarries are removed and sent for SEM analysis.

### Congo Red Assay ---

To visually see the production of curli by bacteria.

##### Materials Required :

1. LB broth
2. Congo Red stain
3. Chloramphenicol antibiotic(2mg/ml)

##### Procedure :

1. Add bacteria in 5mL of culture media
2. Add 50µL of the appropriate selective antibiotic
3. Let bacteria grow at 30°C with a 180 rpm agitation until OD600 = 1
4. Take 5mL of the bacterial culture in a 15mL tubes
5. Pellet down the cells at 13000 rpm for 5 mins
6. Remove the supernatant
7. Add 3mL of the Congo Red solution
8. Let incubate for 10 mins
9. Pellet down the cells at 13000 rpm for 5 mins
10. Remove the supernatant
11. Compare the results with the positive and negative control.

### Biofilm Assay Using Safranin ---

To perform quantification of biofilm formation

##### Materials Required :

1. 96 well plate with clear flat-bottom
2. Overnight cultures
3. 0.1% safranin stain
4. Ethanol
5. 30% acetic acid

##### Procedure :

1. Inoculate a 5ml liquid medium with 5μl 1st Overnight culture, use disposable test tubes and incubate at proper conditions overnight.
2. Add the 2nd overnight culture onto plates and perform the experiment as follows.
3. Add 200μl of diluted 2nd overnight culture into each well (96 well plates) and incubate at proper conditions overnight.
4. Discard the overnight bacteria and wash with Ethanol.
5. Add 200μl 0.1% safranin per well to stain the biofilm.
6. Wait for a couple of minutes, then discard unabsorbed safranin and wash once with ethanol.
7. Add 200 μl of 30% acetic acid into each well and wait for 5 minutes.
8. Measure absorbance of samples at OD of 530 nm.
9. Carefully discard the plate in the biological waste bin after measurement of OD.

### HRP Assay ---

##### Materials Required :

1. 96-well microplates
2. TMB substrate - 3-3'-5-5'-tetramethylbenzidine
3. Horseradish peroxidase.
4. Stop solution (0.16M sulfuric acid)
5. ELISA plate reader.

##### Procedure :

1. Add 100 µL of TMB substrate to each well containing horseradish peroxidase reaction.
2. Incubate at room temperature for 15 to 30 minutes.
3. Add 100 µL of stop solution (0.16M sulfuric acid)to each well.
4. Measure absorbance at 450nm.

### Biofilm Assay Using Crystal Violet ---

##### Experimental Procedure :

The experiments were performed on a 96 well plate.

1. The initial E.coli stock culture was prepared by adding 1000 ul inoculum into 9000 ul standard LB-MES broth with a pH of 7.4 & 5.5 and allowing the E.coli to grow overnight (16-18 hrs) at 37oC and 28oC respectively under orbital shaker at 120 rpm.
2. Next day, the optical density (OD) at 600nm was checked. Value at 600nm must be 2.0 or dilute it.
3. Few wells of plate are filled with 200 ul of fresh LB as blank.
4. The culture was diluted in the ratio of 1:100 with 195 ul of fresh LB - MES broth at a pH of 7.4 and 5ul of culture in the well.
5. The plates were then transferred to thermostatic static incubators maintained at 25℃.
6. To avoid evaporation, the external area of the plate was filled with less amount of distilled water.
7. After 24 hrs, the 96 well plate was taken from the incubator and the planktonic cells were removed.
8. The wells were then washed using sterile PBS.
9. Each well was then added with 200 ul of 0.1% crystal violet and was kept for 15 mins for staining.
10. The crystal violet was then removed and washed with sterile PBS and kept the plate for air drying for 5 mins.
11. The stained biofilm was then solubilised by adding 200 ul of 30% acetic acid.
12. The plate was then kept in a microplate reader and OD value was recorded at 570 nm.

### Cadmium Toxicity Test ---

##### Experimental Procedure :

1. E.coli cells were grown in LB - MES overnight (14 - 16 hrs).
2. Next day, 1% culture suspension was added to freshly prepared LB - MES and incubated at 37oC until OD (600nm) reached 0.5.
3. Then sterile CdCl2 solution was added to various conical flasks with different concentrations (0.18mg/ml),(0.02mg/ml),(0.01mg/ml), all the flasks were then placed in an orbital shaker incubator at 37oC and 130 rpm.
4. Samples were taken at an interval of 30 mins and OD at 600nm was taken spectrophotometrically.
5. Simultaneously viable cell count was done on LB agar from which cell survival was determined.
6. OD vs conc graph is plotted.
7. For checking the morphology of the cells, the cells are subjected to gram staining and are observed under the microscope.

### MTT Assay ---

##### Materials Required :

1. Cell Suspension
2. 96 well plates
3. ELISA reader
4. Hemocytometer

##### Procedure :

Day 1

1. Cells are grown overnight.
2. Cells grown in suspension are harvested by centrifugation at 500 rpm for 5 mins at 2-8o C.
3. Discard the supernatant and resuspend the pellet in 5 ml of sterile PBS. Again centrifuge at 500 rpm for 5 minutes at 2-8o C.
4. Resuspend the pellet in 1ml of PBS.
5. Count the number of cells using hemocytometer and record.
6. Using the sterile PBS serial dilute the cells to 2 fold.
7. Plate the cells at 100 μL per well. Incubate the cells for 6-12 hours at 37o C.

Day 2

1. Add 10 μL of the MTT solution to each well. The Blank contains 10 μL of MTT solution and 100 μL of PBS.
2. Then incubate the plate for 2-4 hours at 37o C.
3. View the cells periodically for the appearance of purple precipitate (intracellular precipitate) using an inverted microscope.
4. Add 100 μL of the solubilization solution to each well and mix thoroughly using a pipette.
5. Leave the plate covered in the dark at 37o C for at least 2 hours. Samples are read after 2 hours.
6. Remove the plate cover and measure the absorbance of the wells, including the blank, at 570 nm.

### Sterilization Methods ---

#### 1. Autoclave

Autoclaving is a process by which high pressure steam is used to clean and sterilize lab equipment and solutions, as well as breakdown nutrient rich tissues that could become biohazard. The steam, which is kept at 121°C, is far above the boiling point of water and is consequently fatal to most microbes and viruses.

##### Materials Required :

1. Glass ware
2. Autoclave
3. Autoclave tray
4. Heat resistant gloves

##### Procedure :

1. Before placing the glassware into the autoclave, ensure enough water has been filled till the required level for the autoclave to function.
2. Place your glass wares into the autoclave after carefully packing them.
3. Using the foot pedlar, close the autoclave machine and tightly screw all the valves.
4. Ensure that the pressure outlet is loosened.
5. Keep the temperature at 121oC for 50 minutes.
6. After completion of autoclave, using the heat resistant gloves, remove the hot glassware from autoclave and place them in the tray.

#### 2. Fumigation

Fumigation is a process where fumigant, a chemical at required temperature and pressure that can exist in a gaseous form in sufficient concentration will be lethal to pest organisms. It is a very useful method to control insect pests, diseases,and nematodes. It is also used for the reduction of microbial agents on surfaces.

##### Materials Required :

1. Personal Protective Equipment set that includes face protection, goggles and mask or face shield, gloves, gown or coverall, head cover, and rubber boots.
2. Formaldehyde solution (37%)
3. Distilled water (D/W)
4. Potassium permanganate
5. Ammonia

##### Procedure :

1. Thoroughly clean windows, doors, floor, walls, and all washable equipment with soap and water.
2. Close windows and ventilators tightly. If any openings are found, seal it with cellophane tape or other material.
3. Switch off all lights, A/C, and other electrical electronic items.
4. Calculate the room size in cubic feet and calculate the required amount of formaldehyde as given in.
5. Adequate care must be taken during wearing PPE that includes face protection, goggles and mask or face shield, gloves, gown or coverall, head cover, and rubber boots.
6. Electric boiler fumigation method for every 1000 cubic feet of the volume of the operation theater-500 ml of 37% formaldehyde added in 1000 ml of water in an electric boiler. Switch on the boiler, leave the room and seal the door.

### References ---

1. <https://www.thermofisher.com/order/catalog/product/N301>
2. Judith H. Merritt,Daniel E. Kadouri, George A. O’Toole. Growing and Analyzing Static Biofilms.2005. <10.1002/9780471729259.mc01b01s00>
3. [http://parts.igem.org/Help:Assembly/3A\_Assembly](http://parts.igem.org/Help%3AAssembly/3A_Assembly)
4. [https://2021.igem.org/Team:IISER-Tirupati\_India](https://2021.igem.org/Team%3AIISER-Tirupati_India)
5. [https://2020.igem.org/Team:Calgary](https://2020.igem.org/Team%3ACalgary)
6. Ya-Wen Chang1, Alexandros A Fragkopoulos1, Samantha M Marquez2, Harold D Kim1, Thomas E Angelini3 and Alberto Fernández-Nieves1.Biofilm formation in geometries with different surface curvature and oxygen availability 6 March 2015.
7. <https://www.addgene.org/protocols/gel-purification/>
8. Pei Yun Lee,1 John Costumbrado,1 Chih-Yuan Hsu,1 and Yong Hoon Kim1.Agarose Gel Electrophoresis for the Separation of DNA Fragments.2012 .doi: 10.3791/3923

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block bnrurl %}https://static.igem.wiki/teams/4509/wiki/banners/results.jpeg {% endblock %}
{% block page\_content %}

#### *"As a man evolves, Nature dissolves"*

Once destroyed, the beauty of nature cannot be reacquired. Humans are causing life on Earth to vanish. Carelessness leads to catastrophic events. When the industrial discharge is released into the water bodies, the heavy metals present in the effluent cause significant damage to the environment, leading to mass disturbances in the terrestrial and marine ecosystems. Cadmium in specific is found to be mutagenic when exposed for a long period of time.

In this page we have shown the results of our recombinant gene expression and reactor model.

Our aim was to produce a solid biofilm structure that would contribute to the production of our hero enzyme, Acid Phosphatase. E.coli K12 strains possess poor biofilm abilities. With the introduction of csgD and OmpR genes, we enhanced the biofilm strength of E.coli K12. OmpR upregulation followed by csgD activation and csgD protein production induced the levels of curli fibers responsible for biofilm formation.Presence of OmpA linker facilitated the cell surface display of acid phosphatase enzyme.

Here, we have proposed an eco-friendly and cost effective solution for the removal of the heavy metal, Cadmium from industrial effluents. We are pleased to inform you that our biologically developed E.coli system has the ability to regulate growth and protein production, thereby increasing the lifespan of Project Curlim!

## Inception of Project CURLIM

---

#### Finalization of Strains

Initially, our team worked with two strains of bacteria, Escherichia coli K12 (wild strain) and Escherichia coli DH5ɑ. We performed characterization for Escherichia coli DH5ɑ by checking it’s growth and color change in EMB agar.

![](https://static.igem.wiki/teams/4509/wiki/results/picture-1.jpg)

![](https://static.igem.wiki/teams/4509/wiki/results/picture-2.jpg)

Figure 1: *Escherichia coli* DH5ɑ in EMB agar

Figure 2: *E.coli* K12 MG1655 in LB agar.

E.coli MG1655 was grown in LB broth and E.coli DH5ɑ was grown in Nutrient broth.We observed the growth curve of K12MG1655 and DH5ɑ.After extensive literature review, we decided to move forward with E.coli K12 MG1655.

![](https://static.igem.wiki/teams/4509/wiki/results/picture-3.png)
Graph 1: Growth curve of *E.Coli* K12 MG1655 and DH5a(OD at 600 nm)

#### Improving the Biofilm formation

To identify the biofilm integrity of the native E.coli, we conducted CV assay. We found that the biofilm formed was not strong enough. The biofilm formation was very poor so we decided to enhance the biofilm formation with the help of our construct BBa\_K4509469.Since it consists of BBa\_J23118 promoter, the burden value is also less and the percentage of growth rate will not be reduced.

![](https://static.igem.wiki/teams/4509/wiki/results/picture-4.jpg)
Figure 3: CV assay forming poor biofilm formation of E.coli MG1655

#### Biofilm Formation in Plasmid-induced biofilm

![](https://static.igem.wiki/teams/4509/wiki/results/cvassay.jpeg)
Figure 4: Crystal Violet assay
![](https://static.igem.wiki/teams/4509/wiki/results/picture-10.png)
Graph 2: OD at 570 nm

## Testing aphA Activity

---

As mentioned earlier,the main element of our project is acid phosphatase enzyme. The following assays were conducted to check the concentration and activity of the enzyme.

#### SDS-PAGE

![](https://static.igem.wiki/teams/4509/wiki/results/sds-results.jpeg)
Figure 5: SDS analysis of Acid Phosphatase

#### Biuret Test

| Particulars | Blank | Standard Solution | | | | | Test | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  | B | S1 | S2 | S3 | S4 | S5 | T1 | T2 |
| Volume of working standard (ml) | - | 0.5 | 1 | 1.5 | 2 | 2.5 | - | - |
| Concentration of Working Standard (mg/ml) | - | 2 | 4 | 6 | 8 | 10 | - | - |
| Volume of Test Solution (ml) | - | - | - | - | - | - | 0.5 | 1 |
| Volume of Distilled Water (ml) | 3 | 2.5 | 2 | 1.5 | 1 | 0.5 | 2.5 | 2 |
| Volume of Biuret solution (ml) | 4 | 4 | 4 | 4 | 4 | 4 | 4 | 4 |
| Optical density at 520 nm | 0 | 0.03 | 0.07 | 0.1 | 0.16 | 0.18 | 0.01 | 0.05 |

![](https://static.igem.wiki/teams/4509/wiki/results/picture-5.jpg)
Figure 6: Biuret's test for quantification of ahpA

#### pNPP Assay

##### 1. With enzyme concentration fixed

| Constant Enzyme | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| Volume of Citrate Buffer (mL) | Volume of pNPP (mL) | Volume of Enzyme (mL) | Volume of Distilled Water (mL) | Volume of NaOH (mL) | OD at 430nm |
| 0.5 | 0 | 0.4 | 0.6 | 2.5 | 0 (Blank) |
| 0.5 | 0 | 0.4 | 0.6 | 2.5 | 0 (Blank) |
| 0.5 | 0.2 | 0.4 | 0.4 | 2.5 | 0.22 |
| 0.5 | 0.3 | 0.4 | 0.3 | 2.5 | 0.27 |
| 0.5 | 0.4 | 0.4 | 0.2 | 2.5 | 0.28 |
| 0.5 | 0.5 | 0.4 | 0.1 | 2.5 | 0.31 |
| 0.5 | 0.6 | 0.4 | 0 | 2.5 | 0.34 |

![](https://static.igem.wiki/teams/4509/wiki/results/picture-6.jpg)
Figure 7: pNPP assay for std enzyme concentration

##### 2. With substrate concentration fixed

| Constant Substrate | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| Volume of Citrate Buffer (mL) | Volume of pNPP (mL) | Volume of Enzyme (mL) | Volume of Distilled Water (mL) | Volume of NaOH (mL) | OD at 430nm |
| 0.5 | 0.4 | 0 | 0.6 | 2.5 | 0 (Blank) |
| 0.5 | 0.4 | 0.1 | 0.5 | 2.5 | 0.1 |
| 0.5 | 0.4 | 0.2 | 0.4 | 2.5 | 0.17 |
| 0.5 | 0.4 | 0.3 | 0.3 | 2.5 | 0.24 |
| 0.5 | 0.4 | 0.4 | 0.2 | 2.5 | 0.25 |
| 0.5 0.4 | 0.5 | 0.1 | 2.5 | 0.29 | |
| 0.5 0.4 | 0.6 | 0 | 2.5 | 0.31 | |

![](https://static.igem.wiki/teams/4509/wiki/results/picture-7.jpg)
Figure 8: pNPP assay for std substrate concentration

## Cadmium Toxicity

---

As we are using cadmium which is a heavy metal, we performed cadmium toxicity for E.coli. We observed the growth curve of the E.coli under different concentrations of the cadmium.

Control → without cadmium

< MIC(Minimum Inhibitory Concentration) → 1 mg/L

MIC → 2.75 mg/L

< Sub-lethal Concentration → 9.18 mg/L

![](https://static.igem.wiki/teams/4509/wiki/results/cd-tox.png)
Graph 3 : Cadmium Toxicity Vs Optical Density (at 600 nm)

#### MTT Assay

The MTT assay is used to measure cellular metabolic activity as an indicator of cell viability, proliferation and cytotoxicity. The cell viability of the cells were observed after 24 hours using 96 well plates.

![](https://static.igem.wiki/teams/4509/wiki/results/picture-21.png)

&ltMIC

MIC

&ltSub-lethal

#### Cadmium-E.coli interaction: Morphology

The morphology of the E.coli K12 strain changed drastically in the presence of Cadmium in sub lethal level (36.68mg/l). Figure 8(a) denotes unexposed cells while Figure 8(b) denotes cells exposed to Cadmium.

![](https://static.igem.wiki/teams/4509/wiki/results/8a.jpeg)

![](https://static.igem.wiki/teams/4509/wiki/results/8b.jpeg)

Figure 9(a)

Figure 9(b)

## Atomic Adsorption Spectroscopy

---

Atomic absorption spectroscopy, is a technique for measuring the concentrations of metallic elements in different materials.Samples were taken at different time intervals to check the concentration of cadmium before the treatment,during the treatment and after the treatment.The result showed significant difference in the concentration of cadmium before and after treatment revealing the efficiency of our project CURLIM!

![](https://static.igem.wiki/teams/4509/wiki/results/atomic-adsorption-spectroscopy.jpeg)
Graph 4 : Atomic Adsorption Spectroscopy

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