Edinburgh/DivisionPopper/Status

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< Edinburgh | DivisionPopper(Difference between revisions)
(→UPDATE)
 
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__TOC__
__TOC__
-
== Plan to model the system: ==
+
== A Proof of Concepts construct: ==
-
 
+
It is always good to move stepwise instead of jumping into the final idea.
It is always good to move stepwise instead of jumping into the final idea.
To show that the region between two ''dif'' sites actually flips every cell division, we started with the proof of concept. The proof of concept involves constructing a construct with a reverse ''lac'' promoter in between two ''dif'' sites with opposite orientation, followed by a YFP gene. Ftsk and Xer were planned to be inserted as it was stated in a paper that extra production of Xer and Fts apart from normal cell production will get better results.
To show that the region between two ''dif'' sites actually flips every cell division, we started with the proof of concept. The proof of concept involves constructing a construct with a reverse ''lac'' promoter in between two ''dif'' sites with opposite orientation, followed by a YFP gene. Ftsk and Xer were planned to be inserted as it was stated in a paper that extra production of Xer and Fts apart from normal cell production will get better results.
The following is the proof of concept design:
The following is the proof of concept design:
-
 
+
<center>
-
[[Image:Proof_of_concept.jpg|600px]]
+
[[Image:Proof1backward.png|500px]]
-
 
+
</center>
== Requirements: ==
== Requirements: ==
-
 
+
* A low copy number plasmid. Ideally only one plasmid should be present.  
-
*1. A low copy number plasmid. Ideally only one plasmid should be present.  
+
* To check the cell changing to yellow colour after flip occurs by means of a fluorescent microscopy, we had to significantly delay the doubling time of the cell.
-
*2. To check the cell changing to yellow colour after flip occurs by means of a fluorescent microscopy, we had to significantly delay the doubling time of the cell.
+
* The fluorescent protein should be degraded completely before the cell division occurs.
-
*3. The fluorescent protein should be degraded completely before the cell division occurs.
+
-
 
+
== Precautions taken to satisfy the requirements: ==
== Precautions taken to satisfy the requirements: ==
 +
* We initially searched for minichromosomes as we found that the oriC was included in the plasmid as a factor to make the plasmid replicate like a chromosome. But the literature search led us to the finding that every cell has 15 copies. So we searched instead for miniF plasmids with very low copy number. We found pHR277 to have a copy number of 1-2. We got the plasmid from Professor Millicent Masters, University of Edinburgh. This plasmid possesses unique XbaI and PstI sites which can be used to insert a biobrick; unfortunately, it also possesses an EcoRI site elsewhere on the plasmid so, unless this is removed, it cannot be converted into a fully compliant vector which can be used to assemble new biobricks
 +
* To delay the doubling time we decided to use a minimal medium which would delay the division to more than an hour.
 +
* In order to account for the fluorescent proteins rapid degradation, we planned to use a fluorescent protein coding sequence with a rapid degradation tag (AANDENYALVA) . This tag with increases the degradation rate brings down the half life of the protein to approximately 40 min (thanks to Dr Drew Endy for the reference paper).
-
*1. We initially searched for minichromosomes as we found that the oriC was included in the plasmid as a factor to make the plasmid replicate like a chromosome. But the literature search led us to the finding that every cell has 15 copies. So we searched instead for miniF plasmids with very low copy number. We found pHR277 to have a copy number of 1-2. We got the plasmid from Professor Millicent Masters, University of Edinburgh. This plasmid possesses unique XbaI and PstI sites which can be used to insert a biobrick; unfortunately, it also possesses an EcoRI site elsewhere on the plasmid so, unless this is removed, it cannot be converted into a fully compliant vector which can be used to assemble new biobricks
+
== miniF plasmid details: ==
-
*2. To delay the doubling time we decided to use a minimal medium which would delay the division to more than an hour.
+
-
*3. In order to account for the fluorescent proteins rapid degradation, we planned to use a fluorescent protein coding sequence with a rapid degradation tag (AANDENYALVA) . This tag with increases the degradation rate to the maximum ( -0.018 1/min) which actually brings down the half life of the protein to approximately 40 min. (Thanks for the paper to Dr.Drew Endy)
+
 +
[[Image:Screenshot.jpg|right]]
 +
{| border="1" cellpadding="10" cellspacing="0"
 +
|-
 +
|Name
 +
|pHR277
 +
|-
 +
|Replicon
 +
|F
 +
|-
 +
|Size
 +
|9.6 kb
 +
|-
 +
|Purpose
 +
|Single Copy Number
 +
|-
 +
|Selectable Marker
 +
|kanamycin
 +
|-
 +
|Other Genes
 +
|lacIQ
 +
|-
 +
|Cloning Site
 +
|ClaI EcoRI HindIII
 +
|-
 +
|Reference
 +
|J. Bacteriol. 179, 5802-5811 (1997)
 +
|-
 +
|Source
 +
|H. Hara
 +
|-
 +
|}
== Construct building: ==
== Construct building: ==
   
   
-
 
*All the necessary parts were taken from the registry. The oligonucleotides for reverse and forward ''dif'' sites (difF and difR) were amplified, combined and biobricked (BBa_I742101 and BBa_I742102).
*All the necessary parts were taken from the registry. The oligonucleotides for reverse and forward ''dif'' sites (difF and difR) were amplified, combined and biobricked (BBa_I742101 and BBa_I742102).
Line 44: Line 72:
*A miniF plasmid (pHR277) was transformed and plated. Primers for FtsK and XerC were ordered and said sequences amplified, digested and biobricked, but only FtsK yielded a final biobrick (BBa_I742164); cells overexpressing XerC refused to grow.
*A miniF plasmid (pHR277) was transformed and plated. Primers for FtsK and XerC were ordered and said sequences amplified, digested and biobricked, but only FtsK yielded a final biobrick (BBa_I742164); cells overexpressing XerC refused to grow.
 +
'''''Primers''''''
 +
{| border="1" cellpadding="20" cellspacing="0"
 +
!Gene
 +
!Forward Primer
 +
!Reverse Primer
 +
|-
 +
|revPlac
 +
|'''revPlacf1:''' tcta gagctc tgtgtgaaattgttatccg
 +
|'''revPlacr1:''' ct actagt a caatacgcaaaccgcctc
 +
|-
 +
|xerCD
 +
|'''xerCD f1:''' tctagagctc cgttggaggattgaacgcg
 +
|'''xerCD r1:''' tactagta ttatttcccccgtttggcg
 +
|-
 +
|ftsK
 +
|'''ftsK f1:''' gc    t  tctagagctc agcctgtaacctggagagc
 +
|'''ftsK r1:''' c t actagt a ttagtcaaacggcggtgg
 +
|-
 +
|YFP
 +
|
 +
|'''YFP r1:''' c t actagt a tataaacgcagaaaggcc
 +
'''YFP r2:''' t actagt a tta t  tataaacgcagaaaggcc
 +
|-
 +
|}
 +
The reverse YFP primer was used in the (failed) assembly of the Proof of Concept device.
-
== General Procedure involved: ==
+
The dif sites were not made by PCR but were synthesised as complementary oligos.
 +
As you will see, there are two pairs of complementary oligos. Each pair was annealed in the PCR machine, digested with SacI and SpeI, and ligated into Edinbrick1.
 +
{| border="1" cellpadding="20" cellspacing="0"
 +
!dif
 +
!oligonucleotides
 +
|-
 +
|dif forward
 +
|'''difF oligo1:''' tcta gagctc ggtg cgca taat gtat atta tgtt aaat t actagtagcg
 +
'''difF oligo2:''' gct actagt a attt aaca taat atac atta tgcg cacc gagctctaga
 +
|-
 +
|dif reverse
 +
|'''difR oligo1:''' tcta gagctc attt aaca taat atac atta tgcg cacc t actagt agcg
 +
'''difR oligo2:''' cgc t actagt a ggtg cgca taat gtat atta tgtt aaat gagctctaga
 +
|-
 +
|}
-
The general procedures involved in the process of biobricking are as follows:
 
 +
== General Procedure involved: ==
 +
 +
The general procedures involved in the process of BioBricking are as follows:
 +
 +
{| width="20%" align="right" style="text-align:center"
 +
|-
 +
|'''Gel'''
 +
|-
 +
|[[Image:P29-1.jpg|right]]
 +
1. ladder (1 kb) 2. plac + YFP 3.plac + YFP (miniprep) 4. XerC 5. ftsk 6.difR + YFP 7. difF + revPlac 8. ladder(1kb).
 +
|}
* Design forward and reverse primers for the sequence of choice
* Design forward and reverse primers for the sequence of choice
Line 56: Line 133:
* Purify the reactions
* Purify the reactions
* Mix cut vector and insert with DNA ligase overnight
* Mix cut vector and insert with DNA ligase overnight
-
* Transform the biobrick into a chemocompetent ''E. coli'' cell line
+
* Transform the BioBrick into a chemocompetent ''E. coli'' cell line
* Plate high and low concentrations of transformed cells onto selective plates and leave overnight
* Plate high and low concentrations of transformed cells onto selective plates and leave overnight
* Replate colonies and inoculate broths overnight with antibiotics that select for the vector
* Replate colonies and inoculate broths overnight with antibiotics that select for the vector
Line 62: Line 139:
* Do analytical restriction digest and/or PCR reactions and run samples on agarose gel to check the insert size
* Do analytical restriction digest and/or PCR reactions and run samples on agarose gel to check the insert size
* Sequence the produced construct.
* Sequence the produced construct.
-
* MaxiPrep correct biobricks.
+
* MaxiPrep correct BioBricks.
It goes without saying that trial-and-error preceeded optimal PCR conditions for each designed primer. We have also employed high-resolution agarose gels when required.
It goes without saying that trial-and-error preceeded optimal PCR conditions for each designed primer. We have also employed high-resolution agarose gels when required.
-
Three attempts to combine difF-PlacRev and difR-YFP have failed to date. We have employed a selection of techniques including gel band excision and purification of both vector and insert and differential restriction enzyme and ligation times. The reason for this repeated failure, using techniques which have worked well for combining other biobricks, is not clear. This project will be continued, using the parts we have developed, by an Honours project student in the laboratory of Dr. Chris French.
 
-
We have now tested the fluorescence of the control construct BBa_I742162 and have shown that we can detect YFP expression.
+
Three attempts to combine difF-PlacRev and difR-YFP have failed to date. We have employed a selection of techniques including gel band excision and purification of both vector and insert and differential restriction enzyme and ligation times. The reason for this repeated failure, using techniques which have worked well for combining other BioBricks, is not clear. This project will be continued, using the parts we have developed, by an Honours project student in the laboratory of Dr. Chris French.
 +
 
 +
 
 +
 
 +
 
 +
We have now tested the fluorescence of the control construct BBa_I742162 and have shown that we can detect YFP expression. The picture below features yesterday's confocal microscopy results.
 +
 
 +
 
 +
 
 +
{| width="20%" align="right" style="text-align:center"
 +
|-
 +
|'''Fluorescence detection'''
 +
|-
 +
|[[Image:Edinburgh_fluo_60x_10s_crop_smaller.jpg]] 
 +
|}
 +
 
 +
==Status==
 +
 
 +
We eagerly anticipate the arrival of the more advanced construct (the actual division PoPper) via GeneArt, but their email correspodence is not promising. The word is that our orders are heavily delayed due to repetitive sequence elements. Some of these are related to the nature of the BioBrick ends, but in this case the problem is exacerbated by the inverse repeat nature of some of the elements. It is now clear that the construct will not be ready before the Jamboree. If it is eventually produced, it will be tested next year by an Honours project student in the laboratory of Dr. Chris French, University of Edinburgh.
 +
 
 +
The laboratory technique protocols can be found at [http://openwetware.org/wiki/French_Lab French Lab - Openwetware]
 +
 
 +
 
 +
 
 +
==UPDATE==
-
[[Image:Edinburgh_fluo_60x_10s_crop_smaller.jpg]]
+
February 2008
-
We're eagerly anticipating the arrival of the more advanced construct (the actual division PoPper) via GeneArt, but their email correspodence is not promising. The word is that our orders are heavily delayed due to repetitive sequence elements. Some of these are related to the nature of the biobrick ends, but in this case the problem is exacerbated by the inverse repeat nature of some of the elements. It is now clear that the construct will not be ready before the Jamboree. If it can eventually be produced, it will be tested next year by an Honours project student in the laboratory of Dr. Chris French, University of Edinburgh.
+
The total Division PoPper construct has arrived and was cloned and introduced into ''E.coli''. We are very pleased to announce that sequencing results show that '''it flips'''. More results will resolve the exact frequency of recombination.
==Biobricks==
==Biobricks==
Line 85: Line 185:
* Jens Bo Andersen, Claus Sternberg, Lars Kongsbak Poulsen, Sara Petersen Bjørn, Michael Givskov,1 and Søren Molin. "New Unstable Variants of Green Fluorescent Protein for Studies of Transient Gene Expression in Bacteria". Appl Environ Microbiol, June 1998, p. 2240-2246, Vol. 64, No. 6.
* Jens Bo Andersen, Claus Sternberg, Lars Kongsbak Poulsen, Sara Petersen Bjørn, Michael Givskov,1 and Søren Molin. "New Unstable Variants of Green Fluorescent Protein for Studies of Transient Gene Expression in Bacteria". Appl Environ Microbiol, June 1998, p. 2240-2246, Vol. 64, No. 6.
 +
* Hiroshi Hara, seiichi Yasuda, Kensuke Horiuchi, James T. Park.A Promoter for the First Nine Genes of the Escherichia coli mra Cluster of Cell Division and Cell Envelope Biosynthesis Genes, Including ftsI and ftsW. Journal Of Bacteriology, Sept. 1997, p. 5802–5811.
 +
* http://gillnet.lab.nig.ac.jp/~cvector/map/ For plasmid image.

Latest revision as of 21:53, 4 March 2008

MENU : Introduction | Background | Applications | Design&Implementation | Modelling | Wet Lab | Synthetic Biology Approach | Conclusions

Contents


A Proof of Concepts construct:

It is always good to move stepwise instead of jumping into the final idea. To show that the region between two dif sites actually flips every cell division, we started with the proof of concept. The proof of concept involves constructing a construct with a reverse lac promoter in between two dif sites with opposite orientation, followed by a YFP gene. Ftsk and Xer were planned to be inserted as it was stated in a paper that extra production of Xer and Fts apart from normal cell production will get better results. The following is the proof of concept design:

Proof1backward.png

Requirements:

  • A low copy number plasmid. Ideally only one plasmid should be present.
  • To check the cell changing to yellow colour after flip occurs by means of a fluorescent microscopy, we had to significantly delay the doubling time of the cell.
  • The fluorescent protein should be degraded completely before the cell division occurs.

Precautions taken to satisfy the requirements:

  • We initially searched for minichromosomes as we found that the oriC was included in the plasmid as a factor to make the plasmid replicate like a chromosome. But the literature search led us to the finding that every cell has 15 copies. So we searched instead for miniF plasmids with very low copy number. We found pHR277 to have a copy number of 1-2. We got the plasmid from Professor Millicent Masters, University of Edinburgh. This plasmid possesses unique XbaI and PstI sites which can be used to insert a biobrick; unfortunately, it also possesses an EcoRI site elsewhere on the plasmid so, unless this is removed, it cannot be converted into a fully compliant vector which can be used to assemble new biobricks
  • To delay the doubling time we decided to use a minimal medium which would delay the division to more than an hour.
  • In order to account for the fluorescent proteins rapid degradation, we planned to use a fluorescent protein coding sequence with a rapid degradation tag (AANDENYALVA) . This tag with increases the degradation rate brings down the half life of the protein to approximately 40 min (thanks to Dr Drew Endy for the reference paper).

miniF plasmid details:

Screenshot.jpg
Name pHR277
Replicon F
Size 9.6 kb
Purpose Single Copy Number
Selectable Marker kanamycin
Other Genes lacIQ
Cloning Site ClaI EcoRI HindIII
Reference J. Bacteriol. 179, 5802-5811 (1997)
Source H. Hara

Construct building:

  • All the necessary parts were taken from the registry. The oligonucleotides for reverse and forward dif sites (difF and difR) were amplified, combined and biobricked (BBa_I742101 and BBa_I742102).
  • Primers for a reverse lac promoter (PlacRev) were ordered and the sequence amplified and biobricked (BBa_I742124).
  • This reverse lac promoter was combined with the dif forward sequence to give the biobrick difF-PlacRev.
  • Yellow fluorescent protein (YFP) (BBa_E0430) was revived from the Registry and combined with the reverse dif site to yield the biobrick difR-YFP, where YFP is the reporter gene.
  • A control construct that consists of the lac promoter and YFP was made for imaging purposes (BBa_I742162).
  • A miniF plasmid (pHR277) was transformed and plated. Primers for FtsK and XerC were ordered and said sequences amplified, digested and biobricked, but only FtsK yielded a final biobrick (BBa_I742164); cells overexpressing XerC refused to grow.

Primers'

Gene Forward Primer Reverse Primer
revPlac revPlacf1: tcta gagctc tgtgtgaaattgttatccg revPlacr1: ct actagt a caatacgcaaaccgcctc
xerCD xerCD f1: tctagagctc cgttggaggattgaacgcg xerCD r1: tactagta ttatttcccccgtttggcg
ftsK ftsK f1: gc t tctagagctc agcctgtaacctggagagc ftsK r1: c t actagt a ttagtcaaacggcggtgg
YFP YFP r1: c t actagt a tataaacgcagaaaggcc

YFP r2: t actagt a tta t tataaacgcagaaaggcc

The reverse YFP primer was used in the (failed) assembly of the Proof of Concept device.

The dif sites were not made by PCR but were synthesised as complementary oligos. As you will see, there are two pairs of complementary oligos. Each pair was annealed in the PCR machine, digested with SacI and SpeI, and ligated into Edinbrick1.

dif oligonucleotides
dif forward difF oligo1: tcta gagctc ggtg cgca taat gtat atta tgtt aaat t actagtagcg

difF oligo2: gct actagt a attt aaca taat atac atta tgcg cacc gagctctaga

dif reverse difR oligo1: tcta gagctc attt aaca taat atac atta tgcg cacc t actagt agcg

difR oligo2: cgc t actagt a ggtg cgca taat gtat atta tgtt aaat gagctctaga


General Procedure involved:

The general procedures involved in the process of BioBricking are as follows:

Gel
P29-1.jpg

1. ladder (1 kb) 2. plac + YFP 3.plac + YFP (miniprep) 4. XerC 5. ftsk 6.difR + YFP 7. difF + revPlac 8. ladder(1kb).

  • Design forward and reverse primers for the sequence of choice
  • PCR amplify the sequence with said primers
  • Double restricion digest the PCR amplified sequence and a vector (Edinbrick1) to create dissimilar sticky ends
  • Purify the reactions
  • Mix cut vector and insert with DNA ligase overnight
  • Transform the BioBrick into a chemocompetent E. coli cell line
  • Plate high and low concentrations of transformed cells onto selective plates and leave overnight
  • Replate colonies and inoculate broths overnight with antibiotics that select for the vector
  • MiniPrep broths that correspond to successful replated colonies
  • Do analytical restriction digest and/or PCR reactions and run samples on agarose gel to check the insert size
  • Sequence the produced construct.
  • MaxiPrep correct BioBricks.

It goes without saying that trial-and-error preceeded optimal PCR conditions for each designed primer. We have also employed high-resolution agarose gels when required.


Three attempts to combine difF-PlacRev and difR-YFP have failed to date. We have employed a selection of techniques including gel band excision and purification of both vector and insert and differential restriction enzyme and ligation times. The reason for this repeated failure, using techniques which have worked well for combining other BioBricks, is not clear. This project will be continued, using the parts we have developed, by an Honours project student in the laboratory of Dr. Chris French.



We have now tested the fluorescence of the control construct BBa_I742162 and have shown that we can detect YFP expression. The picture below features yesterday's confocal microscopy results.


Fluorescence detection
Edinburgh fluo 60x 10s crop smaller.jpg

Status

We eagerly anticipate the arrival of the more advanced construct (the actual division PoPper) via GeneArt, but their email correspodence is not promising. The word is that our orders are heavily delayed due to repetitive sequence elements. Some of these are related to the nature of the BioBrick ends, but in this case the problem is exacerbated by the inverse repeat nature of some of the elements. It is now clear that the construct will not be ready before the Jamboree. If it is eventually produced, it will be tested next year by an Honours project student in the laboratory of Dr. Chris French, University of Edinburgh.

The laboratory technique protocols can be found at [http://openwetware.org/wiki/French_Lab French Lab - Openwetware]


UPDATE

February 2008

The total Division PoPper construct has arrived and was cloned and introduced into E.coli. We are very pleased to announce that sequencing results show that it flips. More results will resolve the exact frequency of recombination.

Biobricks

For the construction of experiments and the Division PoPper several Biobricks are needed. Some of them have been taken from the Registry of Standard Parts and some have been constructed from scratch.

A total of thirteen biobricks were designed, details of which you'll find on the [http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2007&group=Edinburgh Registry team page].


Reference:

  • Jens Bo Andersen, Claus Sternberg, Lars Kongsbak Poulsen, Sara Petersen Bjørn, Michael Givskov,1 and Søren Molin. "New Unstable Variants of Green Fluorescent Protein for Studies of Transient Gene Expression in Bacteria". Appl Environ Microbiol, June 1998, p. 2240-2246, Vol. 64, No. 6.
  • Hiroshi Hara, seiichi Yasuda, Kensuke Horiuchi, James T. Park.A Promoter for the First Nine Genes of the Escherichia coli mra Cluster of Cell Division and Cell Envelope Biosynthesis Genes, Including ftsI and ftsW. Journal Of Bacteriology, Sept. 1997, p. 5802–5811.
  • http://gillnet.lab.nig.ac.jp/~cvector/map/ For plasmid image.
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