Difference between revisions of "Team:IIT-Madras/Description"

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== Experimental Design ==
 
== Experimental Design ==
Noise in any genetic device arises due to various inherent properties of the device. Our study was focused on to address these issues using a device, which was designed to have two genetic components. The first component of the device consisted of the genetic parts (promoters or RBS) required to be characterized were placed in conjunction with a GFP (green florescent protein) producing ORF (open reading frame). Whereas the second component of the device comprised of a RFP (red florescent protein) producing unit placed under fixed promoter. Since the expression level of the second component which consisted of RFP expression would not vary and thus was designed to acts as an internal control. These two genetic components of our device were cloned one after another in the same expression plasmid. The biological parts used in our device for the measurement of inherent noise and how to normalize it to get appropriate results were comprised of different promoters, ribosomal binding sites (RBS), a protein coding parts (ORF) and terminators. Our device was constructed using pSB1A2 plasmid backbone and transformed in E. coli DH5 alpha.
+
Noise in any genetic device arises due to various inherent properties of the device. Our study was focused on to address these issues using a device, which was designed to have two genetic components. The first component of the device consisted of the genetic part (promoter-RBS), required to be characterized, was placed in conjunction with a GFP (green fluorescent protein) producing ORF (open reading frame). Whereas the second component of the device comprised of a RFP (red fluorescent protein) producing unit placed under a fixed promoter-RBS part. Since the expression level of the second component which consisted of RFP expression would not vary and thus was designed to act as an internal control. These two genetic components of our device were cloned one after another in the same expression plasmid. The biological parts used in our device for the measurement of inherent noise and efficient characterization were comprised of different promoters, ribosomal binding sites (RBSs), two protein coding part (GFP, RFP) and terminators. Our device was constructed using pSB1A2 plasmid backbone and transformed in E. coli DH5 alpha cells.
 
   
 
   
To test the role of RBSs, nature of promoters in giving rise to noise, we made six different devices. All of these six devices had same RFP expressing device (consisting of a constitutive promoter, same RBS, red florescent protein ORF) as internal control. Out of six constructed devices, four GFP expressing devices had same IPTG inducible promoter but variation in RBS parts. Whereas in other two device, the GFP expressing component had different constitutive promoters but same RBS.
+
To test the role of RBSs, nature of promoters in giving rise to noise, we made six different devices. All of these six devices had same RFP expressing device. Out of six constructed devices, four GFP expressing devices had same IPTG inducible promoter but variations in RBS parts. Whereas in other two devices, GFP expressing component had different constitutive promoters.
 
====== [[Team:IIT-Madras/Part_Collection|Read more...]] ======
 
====== [[Team:IIT-Madras/Part_Collection|Read more...]] ======
  
 
== Proof ==
 
== Proof ==
We have successfully constructed and cloned all of these devices. We have estimated cumulative intrinsic and extrinsic noise of all the six devices. We have observed a trend that intrinsic noise increases as protein expression from device increases. The obtained noise data provides us a deeper insight into the rise of noise in devices. These experiments helped us to address how we can reduce the impact of these noises in normalization to get accurate result and save us get swayed by erroneous results.  
+
We have successfully constructed and cloned all of these devices. We have estimated cumulative intrinsic and extrinsic noise of all the six devices. We have observed a trend that intrinsic noise increases as protein expression from device increases. The obtained noise data provides us a deeper insight into the rise of noise in devices. These experiments helped us in assessing and understanding the impact of noise, while doing normalization to get accurate results and save us get swayed by erroneous results.  
 
====== [[Team:IIT-Madras/Proof#Noise_in_Devices|Read more...]] ======
 
====== [[Team:IIT-Madras/Proof#Noise_in_Devices|Read more...]] ======
  
 
= RIBOS =
 
= RIBOS =
 
== Design ==
 
== Design ==
In order to make precise and predictable biological devices, which can be controlled at will, we have designed a ribo-regulatory switch and named it RIBOS (RNA Inducible Boolean Output like Switch). RIBOS works on the principle of Watson-Crick base pairing between '''trigger RNA''' and switch mRNA. These RIBOS was placed just before ribosomal binding sites (RBS) in our expression systems, which can be controlled by supplying '''trigger RNA''' molecules. The RIBOS has been designed in such a way that whenever we want expression of the gene placed downstream, we can do that just by supplying the '''trigger RNA'''. The designed RIBOS which leads to expression of the downstream gene has been named RIBOS-ON (or <span class="riboson">RIBOSON</span>). Similarly, we designed another RIBOS using which we can halt the expression the downstream gene at our will. The RIBOS desined by us to halt the expression of the gene under its control has been designated by us as RIBOS-OFF (or <span class="ribosoff">RIBOSOFF</span>). Both these RIBOS devices are great tools in controlling the expression of any gene of interest at our will through ribo-regulation. It has a huge potential applications ranging from detection and quantification of mRNA molecules to the design of independent and modular genetic circuits in limitless number using forward engineering. As a deliverable, the algorithm can be used by future iGEM teams to design <span class="riboson">RIBOSON</span> and <span class="ribosoff">RIBOSOFF</span> for any trigger sequence.
+
In order to make precise and predictable biological devices, which can be controlled at will, we have designed a ribo-regulatory switch and named it '''RIBOS''' ('''R'''NA '''I'''nducible '''B'''oolean '''O'''utput like '''S'''witch). RIBOS works on the principle of Watson-Crick base pairing between '''trigger RNA''' and '''switch mRNA'''. RIBOS has been designed in such a way that whenever we want expression of the gene placed downstream, we just need to supply the '''trigger RNA''' molecules. The designed RIBOS which leads to expression of the downstream gene in the presence of '''trigger RNA''' has been named RIBOS-ON (or <span class="riboson">RIBOSON</span>). Similarly, we designed another RIBOS using which we can halt the expression of the downstream gene at our will. The RIBOS designed by us to halt the expression of the gene under its control has been designated by us as RIBOS-OFF (or <span class="ribosoff">RIBOSOFF</span>). Both of these RIBOS devices are great tools in controlling the expression of any gene of interest at our will through ribo-regulation. It has a huge potential applications ranging from detection and quantification of mRNA molecules to the design of independent and modular genetic circuits in limitless number using forward engineering. As a deliverable, the algorithm can be used by future iGEM teams to design <span class="riboson">RIBOSON</span> and <span class="ribosoff">RIBOSOFF</span> for any trigger sequence.
 
====== [[Team:IIT-Madras/Design|Read more...]] ======
 
====== [[Team:IIT-Madras/Design|Read more...]] ======
  
 
== Proof ==
 
== Proof ==
We have sucssefully cloned '''trigger RNA''' and '''switch RNA''' for <span class="riboson">RIBOSON</span> and <span class="ribosoff">RIBOSOFF</span> devices.  
+
We have successfully cloned '''trigger RNA''' and '''switch RNA''' for <span class="riboson">RIBOSON</span> and <span class="ribosoff">RIBOSOFF</span> devices.  
 
====== [[Team:IIT-Madras/Proof#RIBOS|Read more]] ======
 
====== [[Team:IIT-Madras/Proof#RIBOS|Read more]] ======
  
 
= Notebook =
 
= Notebook =
We started working on our project in April. After ideating with our mentors for a few weeks, we drew up a detailed protocol for our project, and the experiments we would need to do to validate. We also maintained a diary where we noted down all our observations and work done everyday. We had quite an eventful summer - the Indian iGEM team meet up happened in July, and we also came up with the idea for our game 'Codonut' and the GM survey during the same time. By August, most of the project work was done, and we began to work on the game and the GM survey.
+
We started working on our project in April. After ideating with our mentors for a few weeks, we drew up a detailed protocol for our project, and the experiments we would need to do to validate. We also maintained a diary where we noted down all of our observations and work done everyday. We had quite an eventful summer - the Indian iGEM team meet up happened in July, and we also came up with the idea for our game 'Codonut' and the GM survey during the same time. By August, most of the project work was done, and we began to work on the game and the GM survey.
 
====== [[Team:IIT-Madras/Notebook|Read more...]] ======
 
====== [[Team:IIT-Madras/Notebook|Read more...]] ======
  
 
{{IIT-Madras-Bottom/CSS}}
 
{{IIT-Madras-Bottom/CSS}}

Revision as of 19:46, 19 October 2016

Motivation

Few decades from now on, we’ll have synthetic bacteria acting like autonomous robots. They’ll produce bioplastics, food, electricity on their own. These smart designer bacteria will also be able to sense and precisely differentiate cancer cells from normal cells and eliminate them from the affected human body. Keeping this in mind, we wish to make logic based computations in cells as precise and predictive as we have them in our laptop chips. We seek to apply efficient methods and technology to assess, predict and control the variability in cells. Keeping these points in mind, we had designed our project.

If we were to construct a biological oscillator, we would require various genetic parts with different strengths to function like an oscillator. Our oscillator would work only when we make it using parts, which provide complete information about their behavior with respect to other parts. Generally, we use a reporter protein to characterize the functionality of a device and don’t pay much attention to the proper controls, which can lead to erroneous results due to intrinsic and extrinsic factors. Apart from this, we can have variation in device's behavior due to intrinsic and extrinsic factors giving to a badly correlated function. This can be called as noise in the device.

Noise in Devices

Experimental Design

Noise in any genetic device arises due to various inherent properties of the device. Our study was focused on to address these issues using a device, which was designed to have two genetic components. The first component of the device consisted of the genetic part (promoter-RBS), required to be characterized, was placed in conjunction with a GFP (green fluorescent protein) producing ORF (open reading frame). Whereas the second component of the device comprised of a RFP (red fluorescent protein) producing unit placed under a fixed promoter-RBS part. Since the expression level of the second component which consisted of RFP expression would not vary and thus was designed to act as an internal control. These two genetic components of our device were cloned one after another in the same expression plasmid. The biological parts used in our device for the measurement of inherent noise and efficient characterization were comprised of different promoters, ribosomal binding sites (RBSs), two protein coding part (GFP, RFP) and terminators. Our device was constructed using pSB1A2 plasmid backbone and transformed in E. coli DH5 alpha cells.

To test the role of RBSs, nature of promoters in giving rise to noise, we made six different devices. All of these six devices had same RFP expressing device. Out of six constructed devices, four GFP expressing devices had same IPTG inducible promoter but variations in RBS parts. Whereas in other two devices, GFP expressing component had different constitutive promoters.

Read more...

Proof

We have successfully constructed and cloned all of these devices. We have estimated cumulative intrinsic and extrinsic noise of all the six devices. We have observed a trend that intrinsic noise increases as protein expression from device increases. The obtained noise data provides us a deeper insight into the rise of noise in devices. These experiments helped us in assessing and understanding the impact of noise, while doing normalization to get accurate results and save us get swayed by erroneous results.

Read more...

RIBOS

Design

In order to make precise and predictable biological devices, which can be controlled at will, we have designed a ribo-regulatory switch and named it RIBOS (RNA Inducible Boolean Output like Switch). RIBOS works on the principle of Watson-Crick base pairing between trigger RNA and switch mRNA. RIBOS has been designed in such a way that whenever we want expression of the gene placed downstream, we just need to supply the trigger RNA molecules. The designed RIBOS which leads to expression of the downstream gene in the presence of trigger RNA has been named RIBOS-ON (or RIBOSON). Similarly, we designed another RIBOS using which we can halt the expression of the downstream gene at our will. The RIBOS designed by us to halt the expression of the gene under its control has been designated by us as RIBOS-OFF (or RIBOSOFF). Both of these RIBOS devices are great tools in controlling the expression of any gene of interest at our will through ribo-regulation. It has a huge potential applications ranging from detection and quantification of mRNA molecules to the design of independent and modular genetic circuits in limitless number using forward engineering. As a deliverable, the algorithm can be used by future iGEM teams to design RIBOSON and RIBOSOFF for any trigger sequence.

Read more...

Proof

We have successfully cloned trigger RNA and switch RNA for RIBOSON and RIBOSOFF devices.

Read more

Notebook

We started working on our project in April. After ideating with our mentors for a few weeks, we drew up a detailed protocol for our project, and the experiments we would need to do to validate. We also maintained a diary where we noted down all of our observations and work done everyday. We had quite an eventful summer - the Indian iGEM team meet up happened in July, and we also came up with the idea for our game 'Codonut' and the GM survey during the same time. By August, most of the project work was done, and we began to work on the game and the GM survey.

Read more...