Team:DTU-Denmark/BBrick Characterisation

From 2010.igem.org

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<h3>The RNA Polymerase (RNAP)</h3>
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<h2>Introduction</h2>
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<p align="justify">In synthetic biology when creating the minimal cell  XXX factors have been identified as essential for normal function of RNAP, both regarding normal elongation but also in terms of normal termination function. (REFFF) of these XXX have been identified to take part in regulatory function. While the rest is core subunits. Of interest for regulation in terms of termination Anti-termination will be highlighted:</p>
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<p align="justify">Mainly the hard control of the switch is due to a double regulation system build on a both terminator-anti-terminator and repressor anti-repressor regulation. It was out of the scope of this project to construct the entire theoretical developed switch, and characterize the fully constructed switch. Have focused on characterizing the two regulatory systems individually. This was done in order to investigate if the responses were satisfactory to use in a future complete switch construction. (????  By getting the regulatory mechanism of the subparts we further, by modeling, could conclude constraints for successful function of the system and other subparts.
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<ul>
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In this section we describe the design of and the experimental setup used to characterize the subparts of the system and our bio-bricks.</p>
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<li>NusA:</li>
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<h2>Anti-Terminator</h2>
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<li>NusG:</li>
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<p align="justify">Introduction to this part</p>
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<li>NusE:</li>
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<h3>Selecting subparts</h3>
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<li>NusB:</li>
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<p align="justify">Why were these subparts chosen ?<br>
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</ul>
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AIM</p>
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<p align="justify">Due to the construction of the RNAP of many subcomponents and systems, the function of the RNAP can be regulated by only adding or changing one or a few factors. This is the mechanism in the different termination and anti-termination functions described below.</p><br>
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<h3>Design and experimental setup</h3>
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<p align="justify">Figure and table containing normal transcription and normal termination and table with sub-part names and explanation.</p><br>
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<p align="justify">presentation - Figure of setup and explanation</p>
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<h3>Termination</h3>
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<h3>Materials and methods</h3>
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<p align="justify">(terminator introduction)</p>
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<p align="justify">HOW ? what plasmids and why, what measurering method and why?<br>
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<p align="justify">Termination can fall into one of two categories:</p>
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refer to the notebook page with protocols - and actual info from lab.<br>
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<ul>
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<b>mRNA-stability</b><br>
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<li>Intrinsic Termination</li>
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when introducing non-coding sequences problems will acour  with rna-degredation of RNAP is not attracted to the area, to fast degredation, unwanted steam loops. (Reference to the terminator screening plasmids for BB)<br>
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<li>Factor-dependent Termination</li>
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<h3>Results</h3>
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</ul>
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<p align="justify">comments to the results and reference to the BB pages with info and results.</p><br>
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<h4>Intrinsic Termination</h4>
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<p align="justify">Intrinsic Termination can be found to occur at defined template sequences, usually a region of hyphenated inverted sequence symmetry followed by a run of T residues. Termination through intrinsic terminators is stimulated by additional factors, e.g. NusA. Termination occurs due to the stem-loop structure formed by the base-pairing of mRNA with itself caused by inverted sequence symmetry, followed by the run of T residues. The NusA protein causes the RNA-p complex to temporarily stall at the stem-loop structure, when this is followed by a poly-A tail, the RNA-DNA duplex is destabilized. This causes the RNA-p to dissociate from the DNA, thereby terminating transcription. Termination function step-by-step.</p>
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<h4>Factor dependent Termination</h4>
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<p align="justify">Factor-dependent Termination occurs due to events that are not directly related to transcription, such as the release of ribosomes from nascent transcript or DNA damage. One such host termination factor is Rho, which acts on many sites along the bacterial chromosome.
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(( ??MFD, is a host termination factor that is responsible for releasing RNA-p stalled at sites of UV-induced DNA lesions. ??))
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rho-dependent termination is characterized by not having a specific hairpin structure involved in the termination.
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The termination thus happens  whendue to XXXXXX, and what have been found of the termination site any commen sequences or consensus ?????????????????
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the function of rho dependent termination, have been shown to be affected by XXXX.
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The rho binding sites on the mRNA, have been identified from XXbp to XXXbp of stream of the termination site.
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Rho-termination is thus an example of the more complex termination regulation that is not fully understood and can be very difficult to define and use for  engineering purposes. Thus for a more defined anti-termination system the lambda N-protein system and the interaction with the nut-site in the phage genome ????? is a more defined system. Recent research have found out that the Rho termination..? </p>
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<h3>Phages</h3>
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<h2>Repressor function</h2>
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<p align="justify">What do they need to be succesfull? How stable have the function been proved to be. Examples of known phage regulation systems.  Lambda, p21, P22, gifsy1,2,3,. What does the genomes contain of proteins, mRNA, binding sequences,  ANJA??? Identification of mechanisms in regulation, compare with the Chinese paper (hong kong) using cI protein and UV-radiation.
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<p align="justify">Introduction to this part</p>
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Link from phages to anti-terminator and/or Repressor function in phages (next sections).</p>
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<h3>Selecting subparts</h3>
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<h3>Fluorescence Proteins</h3>
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<p align="justify">Why were these subparts chosen ?<br>
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<p align="justify">as we departed in the idea from the terminator screening plasmids described in the partsregistry.org (REFFF) we had a primary focus on fluorescence proteins as our reporter systems. Further we wanted to have high quality data, with a high resolution. We descided on the in-house expertice on using a continous microfermentor system that can measure two fluorescence proteins continuously (biolector) and a flow cytometer, also capable of measuring at two different wave length. </p>
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AIM</p>
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<h3>Design and experimental setup</h3>
 +
<p align="justify">presentation - Figure of setup and explanation</p>
 +
<h3>Materials and methods</h3>
 +
<p align="justify">HOW ? what plasmids and why, what measurering method and why?<br>
 +
refer to the notebook page with protocols - and actual info from lab.<br>
 +
<h3>Results</h3>
 +
<p align="justify">comments to the results and reference to the BB pages with info and results.</p><br>
 +
 
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<h2>Synthetic promoter library (SPL)</h2>
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How does it work, examples, what have it been used to characterize?<br>
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how do you construct it? Figures and illustrations to explain.<br>
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Figures to explain our use? And example on our specific design primer sequences illustration on the double stranded DNA, with BB - prefix suffix.<br></p>
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= Characterizing BBricks as parts of the switch =
 
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'''(Materials and Methods)''' section<br>
 
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Mainly the hard control of the switch is due to a double regulation system build on a both terminator-anti-terminator and repressor anti-repressor regulation. It was out of the scope of this project to construct the entire theoretical developed switch, and characterize the fully constructed switch. Have focused on characterizing the two regulatory systems individually. This was done in order to investigate if the responses were satisfactory to use in a future complete switch construction. (????  By getting the regulatory mechanism of the subparts we further, by modeling, could conclude constraints for successful function of the system and other subparts.
 
-
In this section we describe the design of and the experimental setup used to characterize the subparts of the system and our bio-bricks.
 
-
 
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== Anti-terminator function ==
 
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(experimental work)
 
-
 
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==== selecting subparts ====
 
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Why were these subparts chosen ?<br>
 
-
AIM
 
-
 
-
==== Design and experimental setup ====
 
-
'''presentation - Figure of setup and explanation'''
 
-
 
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==== Materials and methods ====
 
-
HOW ? what plasmids and why, what measurering method and why?
 
-
refer to the notebook page with protocols - and actual info from lab.
 
-
 
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'''mRNA-stability'''
 
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when introducing non-coding sequences problems will acour  with rna-degredation of RNAP is not attracted to the area, to fast degredation, unwanted steam loops. (Reference to the terminator screening plasmids for BB)
 
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'''Synthetic promoter library (SPL)'''<br>
 
-
How does it work, examples, what have it been used to characterize?
 
-
how do you construct it? Figures and illustrations to explain.
 
-
Figures to explain our use? And example on our specific design primer sequences illustration on the double stranded DNA, with BB - prefix suffix.
 
-
 
-
==== Results ====
 
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comments to the results and reference to the BB pages with info and results.
 
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== Repressor function ==
 
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(experimental work)
 
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==== selecting supparts ====
 
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Why were these supparts chosen ?<br>
 
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AIM
 
-
 
-
==== Design and experimental setup ====
 
-
'''presentation - Figure of setup and explanation'''
 
-
 
-
==== Materials and methods ====
 
-
HOW ? what plasmids and why, what measurering method and why?
 
-
refer to the notebook page with protocols - and actual info from lab.
 
-
 
-
==== Results ====
 
-
comments to the results and reference to the BB pages with info and results.
 

Revision as of 12:40, 6 October 2010

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Welcome to the DTU iGEM wiki!

Home The Team The Project Parts submitted Modelling Notebook Blog

Introduction

Mainly the hard control of the switch is due to a double regulation system build on a both terminator-anti-terminator and repressor anti-repressor regulation. It was out of the scope of this project to construct the entire theoretical developed switch, and characterize the fully constructed switch. Have focused on characterizing the two regulatory systems individually. This was done in order to investigate if the responses were satisfactory to use in a future complete switch construction. (???? By getting the regulatory mechanism of the subparts we further, by modeling, could conclude constraints for successful function of the system and other subparts. In this section we describe the design of and the experimental setup used to characterize the subparts of the system and our bio-bricks.

Anti-Terminator

Introduction to this part

Selecting subparts

Why were these subparts chosen ?
AIM

Design and experimental setup

presentation - Figure of setup and explanation

Materials and methods

HOW ? what plasmids and why, what measurering method and why?
refer to the notebook page with protocols - and actual info from lab.
mRNA-stability
when introducing non-coding sequences problems will acour with rna-degredation of RNAP is not attracted to the area, to fast degredation, unwanted steam loops. (Reference to the terminator screening plasmids for BB)

Results

comments to the results and reference to the BB pages with info and results.


Repressor function

Introduction to this part

Selecting subparts

Why were these subparts chosen ?
AIM

Design and experimental setup

presentation - Figure of setup and explanation

Materials and methods

HOW ? what plasmids and why, what measurering method and why?
refer to the notebook page with protocols - and actual info from lab.

Results

comments to the results and reference to the BB pages with info and results.


Synthetic promoter library (SPL)

How does it work, examples, what have it been used to characterize?
how do you construct it? Figures and illustrations to explain.
Figures to explain our use? And example on our specific design primer sequences illustration on the double stranded DNA, with BB - prefix suffix.

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