So far, what do we know about the natural Lac Operon? Well, we know that it is a gene that produces a number of structural proteins, including β-galactosidase. We also know that it requires an absence of glucose and the presence of lactose to function. What we haven't discussed is the function of the operon from an engineering perspective.
[[Image: LacOperonCircuitDiagram.png |thumb|350px||left|A circuit diagram illustrating glucose and lactose as inputs.]]
The Lac Operon is a gene specific to E. Coli that controls the cell's digestion of lactose. It consists of a promoter, an operator, three structural genes, and a terminator. It is both positively and negatively regulated, allowing expression to be contingent on the concentrations of glucose and lactose in the cell.
Structure of the Lac Operon (1)
STRUCTURE
The Lac Operon encodes three structural genes:
LacZ: The Lac Z structural region, or β-galactosidase, hydrolyzes the disaccharide lactose into glucose and galactose, sugars that are smaller and easier for the cell to digest. However, in low concentrations of lactose, β-galactosidase cleaves and rearranges lactose into allolactose, which acts as an inducer for the LacI repressor (see Negative Regulation).
LacY: LacY, or lactose permease, is a transmembrane protein that transports lactose into the cell.
LacA: LacA is a transacetylase. While it has functionality, it has little effect on the function of our design, so it will not be discussed.
In addition to the structural genes, the Lac Operon includes a promoter and an operator region. The promoter region is the area to which the Lac I repressor and the CAP-cAMP complex bind, the mechanics of which will be discussed later (see Positive Regulation and Negative Regulation).
PURPOSE: Efficiency
Expression of the Lac Operon is determined jointly by the levels of glucose and lactose in the cell. Being a monosaccharide, glucose is easier (i.e., takes less energy) to digest; therefore, if glucose is present, the cell will prefer to use it as an energy source. However, if glucose is not available as an energy source, the cell will use lactose instead. A table describing this relationship is below:
Glucose/Lactose Relationship to Lac Operon Transcription (2)
Carbohydrates
CAP-cAMP Complex
LacI Repressor
RNA Polymerase
Transcription of Lac Operon
+ Glucose, + Lactose
Not bound to DNA
Lifted off operator site
Keeps falling off promoter site
Very low transcription
+ Glucose, - Lactose
Not bound to DNA
Bound to operator site
Blocked by repressor
No transcription
- Glucose, - Lactose
Bound to DNA
Bound to operator site
Blocked by the repressor
No transcription
- Glucose, + Lactose
Bound to DNA
Lifted off operator site
Sits on promoter site
TRANSCRIPTION
From this table, we can observe a multitude of things:
Transcription only occurs when lactose, but not glucose, is present.
When glucose is not present, the CAP-cAMP complex (or the activator protein) is not bound to DNA.
Thus, the absence of glucose promotes transcription.
When lactose is not present, the LacI repressor is bound to the operator site. When lactose is absent, the repressor is NOT bound to the operator site.
Thus, the presence of lactose promotes transcription.
For the RNA Polymerase to properly attach to the Lac Operon, the CAP-cAMP complex must be attached to the DNA, and the LacI repressor must not be attached to the operator site.
Therefore, transcription only occurs when lactose, but not glucose, is present.
Why does this phenomenon occur? Well, like stated before, lactose is the cell's last resort energy source because it requires more energy from the cell to digest than does glucose. The enzyme that digests lactose is β-galactosidase, which can only be produced by initiating transcription of the Lac Operon. Thus, to be able to digest lactose, the cell needs to initiate transcription of the Lac Operon.
NEGATIVE REGULATION: The LacI Repressor
Example of negative regulation of the LacI repressor (5)
The genes encoding the LacI repressor are actually located upstream of the Lac Operon. The LacI gene is not regulated; therefore, it is produced continuously. It binds to the Lac Operon in the promoter region; however, it does not bind if there is lactose in the cell. Why is this? Well, the cell produces very low levels of β-galactosidase even when not in the presence of lactose. In these very low lactose conditions, β-galactosidase has a different function: it cleaves lactose and recombines it to form allolactose, which acts as an inducer for LacI. It binds to LacI and causes a conformational change, which in turn makes LacI unable to bind to the promoter region of the Lac Operon.
Positive and negative regulation of the Lac Operon (4)
POSITIVE REGULATION: CAP-cAMP Complex
Remember from before, the absence of the LacI repressor is not the only factor that allows transcription to occur. There is also a form of positive regulation that occurs via the CAP-cAMP Complex, the formation of which is controlled by the levels of glucose within the cell. As glucose levels in the cell begin to decline, E. Coli responds by beginning to synthesize cyclic adenosine monophosphate, or cAMP. As cAMP concentration increases, it binds to a catabolite activator protein, or CAP. cAMP acts as an inducer for CAP, causing a conformational change that allows CAP to bind to the promoter region of the Lac Operon. This cAMP-CAP complex interacts with the RNA polymerase, increasing its affinity for the Lac promoter. Without attachment of the cAMP-CAP complex, or in high levels of glucose, affinity wouldn't be high enough to cause significant transcription.
SUMMARY
So far, what do we know about the natural Lac Operon? Well, we know that it is a gene that produces a number of structural proteins, including β-galactosidase. We also know that it requires an absence of glucose and the presence of lactose to function. What we haven't discussed is the function of the operon from an engineering perspective.
A circuit diagram illustrating glucose and lactose as inputs.
A circuit diagram illustrating glucose and lactose as inputs.
Keep talking about the Lac Operon. Keep talking about the Lac Operon. Keep talking about the Lac Operon. Keep talking about the Lac Operon. Keep talking about the Lac Operon. Keep talking about the Lac Operon. Keep talking about the Lac Operon. Keep talking about the Lac Operon. Keep talking about the Lac Operon. Keep talking about the Lac Operon. Keep talking about the Lac Operon. Keep talking about the Lac Operon. Keep talking about the Lac Operon. Keep talking about the Lac Operon. Keep talking about the Lac Operon. Keep talking about the Lac Operon. Keep talking about the Lac Operon. Keep talking about the Lac Operon. Keep talking about the Lac Operon. Keep talking about the Lac Operon. Keep talking about the Lac Operon. Keep talking about the Lac Operon. Keep talking about the Lac Operon. Keep talking about the Lac Operon. Keep talking about the Lac Operon. Keep talking about the Lac Operon. Keep talking about the Lac Operon. Keep talking about the Lac Operon. Keep talking about the Lac Operon. Keep talking about the Lac Operon. Keep talking about the Lac Operon.
Design: Our genetic circuit
OUR GENE SWITCH:
Device design. Image adapted from [3].
This design creates a simple genetic device that acts as a "sugar switch". It produces a GFP output based on the presence or absence of a sugar input, in this case IPTG.
Our design incorporates BioBrick parts from the Registry of Standard Biological Parts.
Brick 1: IPTG-Inducible Lac Promoter Brick
The first BioBrick includes parts which will work together to create the Lac-I repressor protein, and also includes the promoter which is regulated by it.
This BioBrick consists of:
A consitutive promoter: Causes transcription to begin.
A ribosome binding site: Ribosomes will attach here during transcription.
The gene for the Lac-I repressor protein: This gene is what will be transcribed to create the protein of interest, the Lac-I repressor.
Terminators: End the transcription process.
Lac-I regulated promoter: This promoter will cause the next stage of transcription to begin, and is negatively regulated (repressed) by the Lac-I protein. This means that it will promote the next stage only in the absence of the Lac-I protein.
Brick 2: GFP Production Brick
The second BioBrick includes the parts necessary to produce an output of Green Fluorescence Protein (GFP). This output is regulated by the previous stage.
This BioBrick consists of:
A ribosome binding site: This stage involves a second transcription, so it needs its own site for ribosome binding.
The GFP gene: This is the gene that will be transcribed to use GFP.
Transcription terminators: End the transcription process.
How it Works: The Role of IPTG and Lac-I
The switch response of this device is due to the relationships it creates between IPTG, the Lac-I protein, and the GFP output. Transcription of the GFP output depends on the activity of the stage 2 promoter, the Lac-I regulated promoter. If this promoter is active, GFP will be produced. This promoter is regulated by the Lac-I repressor protein. Presence of the Lac-I protein inhibits the promoter, which turns off GFP production. The Lac-I protein is created in stage 1 of the genetic circuit. In its default state, the reaction would go as follows:
Building: Assembly Scheme
Testing: Modeling and GFP Imaging
A LAC SWITCH MODEL
We used a previously published synthetic switch, developed by Ceroni et al., to understand how our system could potentially be modeled and simulated.
AN INTERACTIVE MODEL
We used a model of the natural Lac operon to understand how changing the parameter values changes the behavior of the system.
COLLECTING IMPERICAL VALUES TO IMPROVE THE MODEL
We explored how one technique, imaging via microscopy could be used to determine the production rate of an output protein, in this case GFP in yeast, could be used to determine a "real" value for maximum GFP production rate under our own laboratory conditions.
Ideally, the GFP production rate measured by this method could be entered as a value for [which parameter] in the Ceroni et al. model.
Human Practices
Danger of Chemicals in Farmlands
Our Team
Shay Ravacchioli
My name is Shay Ravacchioli, and I am a Junior majoring in Biomedical Engineering with minors in Biological Sciences and Psychology. I am taking BME 494 because I think Synthetic Biology is fascinating. An interesting fact about me is that I play piano and guitar.
Jenessa Lancaster
My name is Jenessa Lancaster, and I am a Junior majoring in Biomedical Engineering with a minor in Psychology. I am taking BME 494 because I have always wanted to learn more about Synthetic Biology and Genetic Engineering. An interesting fact about me is that I write songs.
Michael Rose
My name is ###, and I am a ### majoring in ###. I am taking BME 494 because ###. An interesting fact about me is that ###.
Your Name
My name is ###, and I am a ### majoring in ###. I am taking BME 494 because ###. An interesting fact about me is that ###.
Works Cited
[1] Heller, H. Craig., David M. Hillis, Gordon H. Orians, William K. Purves, and David Sadava. Life: The Science of Biology. Sunderland, MA,: Sinauer Ass., W.H. Freeman and, 2008. N. pag. Print.
[2] Escalante, Ananias. "Regulation I." Class Notes. University of Arizona. 20 February 2013.
[3] Registry of Standard Biological Parts. Web. 25 Apr 2013. <partsregistry.org>.
[4] Insert Shay's Microbiology textbook here.
[5] Insert Shay's other Lac Operon image information here.
.jpg)
