Team:Buenos Aires/Results/Bb2

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= Why this biobrick? =
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== Construct Desing based on Biobrick Registry Parts == FALTA MEJORAR ESTA PARTE (MUCHO) , PERO CREO QUE VALE LA PENA PONERLA ==
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We were having some '''custom related''' issues with the synthesized dna we ordered (see [[Team:Buenos_Aires/Results/Bb1 | aa-rich export devices]]), so we started designing '''a biobrick that could be used for the same purpose, but that uses just parts included in the 2012 kit'''. Yes, you are correct: '''a PlanB'''!
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'''His Secretion in Bacteria'''
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Although we did not work in the lab with this ''planB'' biobrick - because fortunately the synthesized dna made it through customs (although very late) - we are including it in the registry and the wiki for documentary purposes only. Just follow this link <partinfo>BBa_K792014</partinfo> to access its entry in the registry.
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In the spirit of the competition we decided to design an extra construct besides our main biobrick, solely by using parts that came at the Biobrick Kit 2012, and that could work for the same purposes that our main biobrick.  
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We designed a plausible construct; however we found several obstacles in each part as we state here.
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= Bacteria Exportable His-rich peptide Generator =
-
From this exercise we can conclude two things:
+
As mentioned before, for this biobrick we had to use solely parts that came in the iGEM Kit 2012 Spring distribution, and that could work for the same purposes that our main biobrick, which is mainly the export of aminoacids.  As there are much more parts for E. coli than for yeast, we decided to build a bacterial device as a "proof of principle" that amino acid secretion can regulated in this way.  
-
- Some biobricks have nOt yet been optimized to be standard and simple to use in one step; it would only be desirable for them to be as easier to use as possible.
+
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- Our main biobrick is an important contribution to the registry part, given that it allows the export of aminoacids to be enhanced with the use of only one part, without the need of the many steps that we describe in this section.
+
-
== Aim ==
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We designed a plausible construct that could work for the export of ''His'' using 5 parts of the registry, but we didn't find enough parts in order to design a simmilar one for the export of ''Trp''.
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To create a biobrick that would enhance Histidine secretion in E. coli using standard parts of the registry as a proof that one can increase the production and secretion of an aminoacid and its measurement in the culture medium.
+
Furthermore, the binding and preparation of this device is much more complex and has many more steps than what our main biobrick would require to work. Therefore, we conclude that our main biobrick is an important contribution to the registry part, given that it allows the export of aminoacids His and Trp to be enhanced with the use of only one part, without the need of the many steps that we describe in this section and consequently reducing the risk of failure and errors.
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To learn how to use and merge standard parts from the registry provided at the iGEM Kit.
+
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To characterize the functioning of existent parts in the registry and new combinations of them, therefore contributing the improvement of the iGEM record.  
+
 +
<!--
 +
==== Aim ====
-
== Parts to use ==
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* To create a biobrick that would enhance Histidine secretion in E. coli using standard parts of the registry as a proof that one can increase the production and secretion of an aminoacid and its measurement in the culture medium.
 +
* To learn how to use and merge standard parts from the registry provided at the iGEM Kit.
 +
* To characterize the functioning of existent parts in the registry and new combinations of them, therefore contributing the improvement of the iGEM record.
 +
* To proove that our main biobricks, devices 1 and 3 for the export of His, are an important contribution to iGEM, given that they are capable of His export with far less steps involved.
 +
* To proove that our main biobricks, devices 2 and 4 for the export of Trp, are an important contribution to iGEM, given that there are was no biobrick with such function available.
 +
-->
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Promoter (constitutive or inducible); RBS; Peptide Signal (Secretion Tag), Histitide Tag; Terminator.
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== Device final structure ==
 +
{|
 +
|
 +
Our device is composed by:
 +
* '''Promoter''' (<partinfo>BBa_J04500</partinfo>)
 +
* '''Ribosome binding site''' (<partinfo>BBa_B0034</partinfo>)
 +
* '''Peptide signal''' or secretion tag so that the aminoacid is exported out of the cell (<partinfo>BBa_K125310</partinfo>)
 +
* '''Payload''', a ''histidine tag'' repeated several times in order to have a long peptide enriched in this aa (<partinfo>BBa_K133035</partinfo>)
 +
* '''Terminator''' (<partinfo>BBa_B0024</partinfo>)
 +
|
 +
{|width="100%"
 +
|+ '''Structure of the device'''
 +
|align="center" | [[File:Bsas2012-Bb5.png|500px]]
 +
|}
 +
|}
 +
This construct should be able to export '''Histidine''', for sure in ''Cyanobacterium'' and to be tested in ''E.Coli'' and ''S. cereviciae''.
 +
'''More details about the design process (and choices made) can be found in the following sections'''.
 +
== Design process ==
 +
=== Promoter ===
 +
We found many usable parts to use as promoters. We found Promoters + RBS ideal for our purposes, in order to economize ligation steps.
 +
We considered using two parts:
-
== Promoter choice ==
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# Promoter + RBS (<partinfo>BBa_K206015</partinfo>), a strongest constitutive promoter in J23100 family (J23100) + mid-strength RBS from the community collection (B0030, 0.6.  It looks like a reliable sequence but it has not been tested according to the registry.
 +
# Inducible Promoter (IPTG) + RBS (Strong)  (<partinfo>BBa_J04500</partinfo>). This part has been tested and according to the registry it works well.
-
We found many usable parts to use as promoters and chose:
+
We finally decided to use the second opton (<partinfo>BBa_J04500</partinfo>)  in order to make our system plausible of regulation through IPTG. This kind of regulation could also have been implemented in our main biobricks, for the same purposes or making the system more flexible.
 +
=== Signal Peptide  ===
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Promoter + RBS: BBa_K081005
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Unfortunately, we found very few signal peptide biobrick options, solely two and tested in Cyanobacterium. Our two options were:
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http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100
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http://partsregistry.org/wiki/index.php?title=Part:BBa_B0030
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Inducible Promoter (IPTG) + RBS (Strong): BBa_J04500
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# pilA1 signal sequence from cyanobacterium Synechocystis; secretes protein: <partinfo>BBa_K125300</partinfo>
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http://partsregistry.org/wiki/index.php?title=Part:BBa_R0010
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# slr2016 signal sequence from cyanobacterium Synechocystis; secretes protein: <partinfo>BBa_K125310</partinfo>
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http://partsregistry.org/wiki/index.php?title=Part:BBa_B0034
+
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We would use the second one, so that the system is plausible of regulation.  
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These parts are only partically confirmed and optimized for working in Cyanobacterium, not E. coli or Yeast. We could use any of them in order to test them but not having any E.coli or yeast optimized signal peptide available at the registry is a critical obstacle in the project.
-
 
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 +
We would use the second one:  <partinfo>BBa_K125310</partinfo>.
 +
=== Payload ===
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== Signal Peptide Options ==
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The only option available in iGEM Kit Spring distribution 2012 was Methionine + His Affinity Tag x 6: <partinfo>BBa_K133035</partinfo>. This part is only partially confirmed. We would put this part 3 times in a row in order to have a larger peptide - so it would take 3 ligation steps to obtain a larger peptide enriched Histidine, whereas in our main biobrick 1 and 3, it already comes in the same construct.
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Unfortunately, we found very few signal peptide biobrick options, solely two and tested for cyanobacterium.  
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"pilA1 signal sequence from cyanobacterium Synechocystis; secretes protein": BBa_K125300 (partially confirmed)
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=== Terminator ===
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http://partsregistry.org/Part:BBa_K125300
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{|
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tggctagtaattttaaattcaaactcctctctcaactctccaaaaaacgggcagaaggtggt
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|- valign="top"
 +
| width="70%" |
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"slr2016 signal sequence from cyanobacterium Synechocystis; secretes protein" :  BBa_K125310 (partially confirmed)
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There are several options for terminators, but we would use a double terminator in order to be sure that it works. We choose the double terminator <partinfo>BBa_B0024</partinfo>. This sequence has a double terminator in several of reading frames, which could be very useful.
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http://partsregistry.org/Part:BBa_K125310
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| align="center" |
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tggcagcaaaacaactatggaaaattttcaatcctagaccgatgaagggtgga
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{| class="wikitable" style="width:200px"
 +
  |-
 +
|<!--column1-->[[File:Bsas2012-Translate.png|200px]]
 +
|-
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|Different Reading Frames of BBa_B0024. Frames 1 and 3 are useful as double terminators.
 +
|}
 +
|}
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We could use any of them, but knowing that they are fit for Cyanobactierium, not E. Coli.
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== Assembly method ==
 +
All the chosen parts are compatible with '''RFC 21 Standard''', which is an in frame assembly method and would be our choice for assembling these parts.
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{| class="wikitable" border="1"
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== HisTag Options ==
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|-
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  |<!--column1-->[[File:Bsas2012-Finalbb5.JPG|500px]]
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|-
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Methionine + His Affinity Tag x 6: BBa_K133035 (partially confirmed)
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|Picture: Final sequence with prefix, suffix and scars assembled with RFC 21 Standard method.  
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http://partsregistry.org/Part:BBa_K133035
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  |}
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http://partsregistry.org/cgi/partsdb/puttext.cgi
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-
 
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Methionine is there only to be able to express the protein, so this is a HisTag, and it is functional to our purposes.
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We would put this 3 times in a row to make the histag stronger but then we have 2 questions:
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-
 
+
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- the methionine that remains in the middle of each histag, would it make the structure unstable?
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For example, placing it three times in a row we get:  
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atgcaccaccaccaccaccac/atgcaccaccaccaccaccac/atgcaccaccaccaccaccac
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-
 
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- This sequence does not carry a Stop Codon and we found that several of the parts of the registry do not carry one either, which could be a major issue.
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-
 
+
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His Tag BBa_K157011 (Bad Sequencing :( )
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http://partsregistry.org/Part:BBa_K157011:Design
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Discarded for Bad Sequencing
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-
 
+
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== Stop Codon ==
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-
 
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There are no stop codons in the HIsTag availables at the Kit. Therefore we would need to use another biobrick carrying a stop codon and use its restriction sites to cut it and keep only the Stop Codon part.
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-
 
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There is a biobrick that has several things and then ends with a hisstop. It has several restriction sites before a 6His and Stop, so we could use this part as a biobrick if we were able to cut it.  
+
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http://partsregistry.org/Part:BBa_K133038
+
-
 
+
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In this site we can see the restriction sites present at this construct:
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http://tools.neb.com/NEBcutter2/cutshow.php?name=c8ecf24a-
+
-
 
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What restriction enzyme do we use in order for the remaining part to be used as a biobrick together with the other ones?
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-
 
+
-
 
+
-
 
+
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== Terminator ==
+
-
There are several options for terminators
+
-
 
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Doble terminador: BBa_B0015 ;  BBa_B0024
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-
 
+
-
From Coliphage:  BBa_B0012
+
-
 
+
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Any of them would be ok for our purposes.
+

Latest revision as of 02:55, 27 September 2012

Contents

Why this biobrick?

We were having some custom related issues with the synthesized dna we ordered (see aa-rich export devices), so we started designing a biobrick that could be used for the same purpose, but that uses just parts included in the 2012 kit. Yes, you are correct: a PlanB!

Although we did not work in the lab with this planB biobrick - because fortunately the synthesized dna made it through customs (although very late) - we are including it in the registry and the wiki for documentary purposes only. Just follow this link <partinfo>BBa_K792014</partinfo> to access its entry in the registry.

Bacteria Exportable His-rich peptide Generator

As mentioned before, for this biobrick we had to use solely parts that came in the iGEM Kit 2012 Spring distribution, and that could work for the same purposes that our main biobrick, which is mainly the export of aminoacids. As there are much more parts for E. coli than for yeast, we decided to build a bacterial device as a "proof of principle" that amino acid secretion can regulated in this way.

We designed a plausible construct that could work for the export of His using 5 parts of the registry, but we didn't find enough parts in order to design a simmilar one for the export of Trp.

Furthermore, the binding and preparation of this device is much more complex and has many more steps than what our main biobrick would require to work. Therefore, we conclude that our main biobrick is an important contribution to the registry part, given that it allows the export of aminoacids His and Trp to be enhanced with the use of only one part, without the need of the many steps that we describe in this section and consequently reducing the risk of failure and errors.


Device final structure

Our device is composed by:

  • Promoter (<partinfo>BBa_J04500</partinfo>)
  • Ribosome binding site (<partinfo>BBa_B0034</partinfo>)
  • Peptide signal or secretion tag so that the aminoacid is exported out of the cell (<partinfo>BBa_K125310</partinfo>)
  • Payload, a histidine tag repeated several times in order to have a long peptide enriched in this aa (<partinfo>BBa_K133035</partinfo>)
  • Terminator (<partinfo>BBa_B0024</partinfo>)
Structure of the device
Bsas2012-Bb5.png

This construct should be able to export Histidine, for sure in Cyanobacterium and to be tested in E.Coli and S. cereviciae. More details about the design process (and choices made) can be found in the following sections.

Design process

Promoter

We found many usable parts to use as promoters. We found Promoters + RBS ideal for our purposes, in order to economize ligation steps. We considered using two parts:

  1. Promoter + RBS (<partinfo>BBa_K206015</partinfo>), a strongest constitutive promoter in J23100 family (J23100) + mid-strength RBS from the community collection (B0030, 0.6. It looks like a reliable sequence but it has not been tested according to the registry.
  2. Inducible Promoter (IPTG) + RBS (Strong) (<partinfo>BBa_J04500</partinfo>). This part has been tested and according to the registry it works well.

We finally decided to use the second opton (<partinfo>BBa_J04500</partinfo>) in order to make our system plausible of regulation through IPTG. This kind of regulation could also have been implemented in our main biobricks, for the same purposes or making the system more flexible.

Signal Peptide

Unfortunately, we found very few signal peptide biobrick options, solely two and tested in Cyanobacterium. Our two options were:

  1. pilA1 signal sequence from cyanobacterium Synechocystis; secretes protein: <partinfo>BBa_K125300</partinfo>
  2. slr2016 signal sequence from cyanobacterium Synechocystis; secretes protein: <partinfo>BBa_K125310</partinfo>

These parts are only partically confirmed and optimized for working in Cyanobacterium, not E. coli or Yeast. We could use any of them in order to test them but not having any E.coli or yeast optimized signal peptide available at the registry is a critical obstacle in the project.

We would use the second one: <partinfo>BBa_K125310</partinfo>.

Payload

The only option available in iGEM Kit Spring distribution 2012 was Methionine + His Affinity Tag x 6: <partinfo>BBa_K133035</partinfo>. This part is only partially confirmed. We would put this part 3 times in a row in order to have a larger peptide - so it would take 3 ligation steps to obtain a larger peptide enriched Histidine, whereas in our main biobrick 1 and 3, it already comes in the same construct.

Terminator

There are several options for terminators, but we would use a double terminator in order to be sure that it works. We choose the double terminator <partinfo>BBa_B0024</partinfo>. This sequence has a double terminator in several of reading frames, which could be very useful.

Bsas2012-Translate.png
Different Reading Frames of BBa_B0024. Frames 1 and 3 are useful as double terminators.

Assembly method

All the chosen parts are compatible with RFC 21 Standard, which is an in frame assembly method and would be our choice for assembling these parts.

Bsas2012-Finalbb5.JPG
Picture: Final sequence with prefix, suffix and scars assembled with RFC 21 Standard method.
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