Team:DTU-Denmark/Project testing sRNA

From 2011.igem.org

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(Results and Conclusions)
 
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== Motivation ==
== Motivation ==
-
The biological experiments of the project is a proof of concept, showing that the ''E. coli'' sRNA ''sroB''  and the intergenic sRNA (trap-RNA) in the ''chbBCARG'' gene can be used to control gene expression by targeting the ''ybfM'' Shine-Dalgarno, and that these sRNAs can be rationally designed to target other Shine-Dalgarno sequences.
+
The biological experiments of the testing sRNA part is a proof of concept, showing that the ''E. coli'' sRNA ''sroB''  and the intergenic sRNA (trap-RNA) in the ''chbBCARG'' gene can be used to control gene expression by targeting the ''ybfM'' Shine-Dalgarno, and that these sRNAs can be rationally designed to target other Shine-Dalgarno sequences. The purpose is two-fold to construct plasmids and strains with deletions.
-
The experimental part consists of 3 distinct areas:
+
'''Strain construction''' involves deleting the original genes from the chromosome of a ''E. coli'' W3110 strain. Since we use the original genes, these need to be deleted from the chromosome to prevent them from interfering with our measurements.
-
 
+
-
* Construction of plasmids
+
-
* Strain construction
+
-
* Improving the ''araBAD'' promoter
+
'''Construction of plasmids''' necessary for testing our system involves taking the native system from ''E. coli'', as well as a slightly modified system and putting them into plasmids that let us both control the expression of these components and measure the output of the system.
'''Construction of plasmids''' necessary for testing our system involves taking the native system from ''E. coli'', as well as a slightly modified system and putting them into plasmids that let us both control the expression of these components and measure the output of the system.
-
 
-
'''Strain construction''' involves deleting the original genes from the chromosome of a ''E. coli''  W3110 strain. Since we use the original genes, these need to be deleted from the chromosome to prevent them from interfering with our measurements.
 
-
 
-
'''Improving the ''araBAD'' promoter''' entails expanding the dynamic range of this promoter by modifying the -10 and -35 sequence of the promoter, as well as randomly changing the nucleotide sequence around and in between these sequences. Since the ''araBAD'' promoter is used in our project improving this promoter could lead to even finer control of our system.
 
-
 
-
== Construction of plasmids ==
 
-
 
-
In order to quantitatively describe the natural trap-RNA system the following three plasmids were constructed:
 
-
 
-
*P<span class="subscript">chiP</span>-lacZ plasmid
 
-
: To mimic and test repression of the chiP gene its upstream region of 599 bp was fused to reporter genes lacZ and inserted on the pSB1C3 plasmid. Expression from P<span class="subscript">chiP</span> promoter is costitutive however it can be induced to a higher level by chitobiose. LacZ codes for $\beta$-galactosidase and its activity was tested using $\beta$-Gal assay.
 
-
 
-
*P<span class="subscript">tet</span>-chiX plasmid
 
-
: ChiX, which is 84 bp long, was inserted into pSB4K plasmid together with regulatory element from ''tet'' operon. Expression from P<span class="subscript">tet</span> promoter was induced using anhydrotetracycline.
 
-
 
-
*P<span class="subscript">BAD</span>-chiXR plasmid
 
-
: Introgenic region from chbBCARG operon 284 bp long was inserted into pSB3T5 plasmid. Expression from that plasmid was controlled by regulatory element from ''ara'' operon with arabinose as inducer.
 
-
 
-
Details on how the plasmids were constructed, including information on primers used, can be found in [https://2011.igem.org/Team:DTU-Denmark/Technical_stuff_lab labnotebook].
 
-
 
-
[[File:DTU1_Plasmids.png|300px|thumb|center|add some text]]
 
== Strain construction ==
== Strain construction ==
-
The strain used in testing our system is based on two strains: IG9 a ''E. coli'' based on the wild-type ''Escherichia coli'' K-12<span class="superscript">[[#References|[3]]]</span> strain W3110 with the ''lacZYA'', ''chbBCARG'' operons deleted, and IG302 which is the K-12 ''$Delta$lacZYA'', ''$Delta$chiP'' (alias ''ybfM''), ''$Delta$chiX'' (alias ''sroB'', ''micM'') strain constructed by Rasmussen et al.<span class="superscript">[[#References|[12]]]</span>.  
+
The strains used in testing our system were: '''IG9''' a ''E. coli'' based on the wild-type K-12<span class="superscript">[[#References|[3]]]</span> strain W3110 with the ''lacZYA'', ''chbBCARG'' operons deleted, and '''IG302''' which is the K-12 ''$\Delta$lacZYA'', ''$\Delta$chiP'' (alias ''ybfM''), ''$\Delta$chiX'' (alias ''sroB'', ''micM'') strain constructed by Rasmussen et al.<span class="superscript">[[#References|[6]]]</span>.  
-
 
+
''chbBCARG'', ''chiP'' and ''chiX'' code for utilization of chitobiose (a sugar), chitobiose permease and the regulation of the chitobiose permease respectively.
-
The three latter code for utilization of chitobiose (a sugar), chitobiose permease and the regulation of the chitobiose permease respectively.
+
We used recombineering<span class="superscript">[[#References|[1]]]</span> to delete these genes. Schematically the procedure works in the following manner:
We used recombineering<span class="superscript">[[#References|[1]]]</span> to delete these genes. Schematically the procedure works in the following manner:
Line 58: Line 32:
An example of a linear piece of DNA that could be used with this method, as well as the simplified representation of the procedure, can be seen in figure:
An example of a linear piece of DNA that could be used with this method, as well as the simplified representation of the procedure, can be seen in figure:
-
[[File:DTU1_Redswap.png|665px|thumb|center|add some text]]
+
[[File:DTU1_Redswap.png|400px|thumb|right|A schematic overview of the Recombineering procedure. A linear piece of DNA, carrying a resistance marker is transformed into a strain carrying two helper plasmids, the $\lambda{}$ phage recombination system mediates integration of the linear piece of DNA. The Cre recombinase mediates the excision of the resistance marker.]]
 +
[[File:DTU1_Lox_sites.png|400px|thumb|right|The Cre recombinase recognizes the lox-sites. The mutated sites lox-71 and lox-66 are recognized, but the resulting lox-site is not.]]
 +
In case of two or more subsequent gene deletions using Red-Cre recombineering a problem of recombination between an old ''loxP'' site and newly introduced ones arises. Remember, that after each round of redswap one ''loxP'' site is left in the genome. As a result recombination using Cre recombinase in second or next redswap might not occur between loxP sites flanking a resistance gene. To circumvent this problem two mutated loxP sequences are used - ''lox66'' and ''lox71''<span class="superscript">[[#References|[4]]]</span>. In spite of the mutations these lox sites are recognized by Cre which can remove the intervening region. Finally, from ''lox66'' and ''lox71'' a new site is formed, ''lox72'', which has a reduced affinity to Cre recombinase. Thus, more than one gene deletions can be subsequently conducted.
-
[[File:DTU1_Lox_sites.png|400px|thumb|right|add some text]]
+
== Construction of plasmids ==
-
In case of two or more subsequent gene deletions using Red-Cre recombineering a problem of recombination between an old ''loxP'' site and newly introduced ones arises. Remember, that after each round of redswap one ''loxP'' site is left in the genome. As a result recombination using Cre recombinase in second or next redswap might not occur between loxP sites flanking a resistance gene. To circumvent this problem two mutated loxP sequences are used - ''lox66'' and ''lox71''<span class="superscript">[[#References|[4]]]</span>. In spite of the mutations these lox sites are recognized by Cre which can remove the intervening region. Finally, from lox66 and lox71 a new site is formed, ''lox72'', which has a reduced affinity to Cre recombinase. Thus, more than one gene deletions can be subsequently conducted.
+
-
== Improving the ''araBAD'' promoter ==
+
In order to quantitatively describe the natural trap-RNA system the following four plasmids were constructed:
-
=== ''araBAD'' promoter ===
+
*P<span class="subscript">chiP</span>-lacZ plasmid
 +
: To mimic and test repression of the chiP gene its upstream region of 599 bp was fused to reporter genes lacZ and inserted on the pSB1C3 plasmid. Expression from P<span class="subscript">chiP</span> promoter is costitutive however it can be induced to a higher level by chitobiose. LacZ codes for $\beta$-galactosidase and its activity was tested using $\beta$-Gal assays.
-
In our project to control sRNA chiXR expression we used the arabinose promoter (P<span class="subscript">BAD</span>) from the ''araBAD'' operon that is regulated by araC protein<span class="superscript">[[#References|[10]]]</span>. AraC can work both as a repressor and activator of the PBAD and the mode of action depends on the presence of arabinose. P<span class="subscript">BAD</span> promoter becomes activated in the presence of arabinose and repressed when arabinose is unavailable.
+
*P<span class="subscript">chiP</span>(mutated)-lacZ plasmid
 +
: This plasmid is similar to the P<sub>chiP</sub>-lacZ plasmid, except the Shine-Dalgarno has been changed. This is expected to abolish or limit the regulatory effect of chiX.
-
Choice of P<span class="subscript">BAD</span> was not accidental. AraC repression in the absence of arabinose provides a very low level of background expression which can be lowered even more by adding glucose (catabolic repression). Once induced, this promoter allows a moderate level of expression. Finally, a range of arabinose concentrations can be used to achieve a desired level of induction<span class="superscript">[[#References|[7]]]</span><span class="superscript">[[#References|[10]]]</span>.  
+
*P<span class="subscript">tet</span>-chiX plasmid
 +
[[File:DTU1_Plasmids.png|300px|thumb|right|A schematic representation of the constructed plasmids, and the backbones we placed them in.]]
 +
: ChiX which is 84 bp long and regulates the wild-type chiP Shine-Dalgarno<span class="superscript">[[#References|[2]]]</span><span class="superscript">[[#References|[5]]]</span>, was inserted into pSB4K under the control of the inducible promoter P<sub>tet</sub>. Expression from P<span class="subscript">tet</span> promoter was induced using anhydrotetracycline.
-
There are however some known issues with P<span class="subscript">BAD</span> promoter. Heterogeneous induction, known also as “all-or-nothing”<span class="superscript">[[#References|[6]]]</span> induction of cell culture is one of them. As a result only a subset of cells becomes induced with the arabinose. To circumvent “all-or-nothing” problem the arabinose transporter (AraE) could be placed under inducible promoter<span class="superscript">[[#References|[7]]]</span> or a different transporter could be engineered to have affinity to arabinose, e.g. LacY<span class="superscript">[[#References|[11]]]</span>, and facilitates its uptake. Another problem arises when one wants to use P<span class="subscript">BAD</span> promoter along with another promoter, e.g. P<span class="subscript">prpB</span> promoter from propionate operon, and achieve a similar level of induction from both of them. This might not be feasible as P<span class="subscript">BAD</span> is considerably weaker in expression than aforementioned PprpB promoter<span class="superscript">[[#References|[8]]]</span><span class="superscript">[[#References|[10]]]</span>.
+
*P<span class="subscript">BAD</span>-chiXR plasmid
 +
:A 284 bp long introgenic region from chbBCARG operon known to regulate chiX<span class="superscript">[[#References|[2]]]</span><span class="superscript">[[#References|[5]]]</span> was inserted into pSB3T5 plasmid. Expression from that plasmid was controlled by regulatory element from ''ara'' operon with arabinose as inducer.
-
=== Approach ===
+
Details on how the plasmids were constructed, including information on primers used, can be found in [https://2011.igem.org/Team:DTU-Denmark/Notebook lab notebook].
-
Since control of the system is essential to the applicability of our system we attempted to modify the dynamic range of the arabinose promoter ([http://partsregistry.org/Part:BBa_I0500 BBa_I0500]) by two means:
+
==== Verifying constructs ====
-
*rationally designing the -10 and -35 sequences of the promoter region since both the -35 (CTGACG) and the -10 (TACTGT) of the arabinose promoter are far from wild-type (TTGACA and TATAAT respectively).
+
The plasmids we constructed were verified in two ways. The P<sub>BAD</sub>-chiXR fusion and P<sub>chiP</sub>-lacZ fusion were tested by restriction analysis of the plasmid. And the P<sub>tet</sub>-chiX fusion was tested by PCR.
-
*changing the nucleotides around and between the -10 and the -35 using synthetic promoter library (SPL), without changing the -10 and -35 from wildtype.
+
-
The arabinose promoter we were improving was fused to GFP and this construct came as a biobrick [http://partsregistry.org/Part:BBa_I746908 BBa_I746908]. To introduce the changes a set of primers was used to PCR P<sub>BAD</sub>. Four primers, SRS56 from 1 to 4, were used to make PCR products with mutated nucleotides within -10 and -35 and SRS56SPL primer was used to make the SPL PCR products. Then a round of restriction digestions and ligations followed which allowed to exchange the wild type P<sub>BAD</sub> with the mutated ones. Then they were transformed into E. coli strain DH5$\alpha$. Finally, all the rationally changed P<sub>BAD</sub> promoters and randomly selected SPL mutants were characterized using Biolektor.
+
The restriction of three plasmids tested revealed fragments of the expected sizes. These can be seen in the table below. The gel picture is also available.
-
Detailed description of the experiments, including information on primers used, can be found in [https://2011.igem.org/Team:DTU-Denmark/Technical_stuff_lab labnotebook].
 
-
[[File:DTU1_Arabinose_promoter.png|665px|thumb|right|add some text]]
+
{| class="wikitable" | center
 +
|+P<sub>BAD</sub>-chiXR
 +
|-
 +
! Restriction Enzymes
 +
! Expected sizes
 +
|-
 +
| EcoRI + BamHI
 +
| 1150 + 2550
 +
|-
 +
| EcoRI + NcoI
 +
| 2930 + 770
 +
|}
-
=== Results ===
+
[[File:DTU1_ChiXR_verification.png|665px|thumb|center| A linearized version of the P<sub>BAD</sub>-chiXR construct inside the pSB1C3 plasmid. Restriction sites used, as well as expected fragment sizes are indicated.]]
-
All 4 rationally designed (called Strain 1b, 2a, 3h, 4d) and 18 SPL-changed arabinose promoters (called SPL-1 up to SPL-24) fused to GFP were tested under induction of arabinose at concentration 0.2% and compared to non-induced state. Also, the original [http://partsregistry.org/Part:BBa_I746908 BBa_I746908] was characterized in the same fashion.
+
[[File:DTU-ChiXR.png|370px|thumb|left| The gel picture of the P<sub>BAD</sub>-chiXR construct. The restriction enzymes used, and the sizes are indicated on the picture. EN = EcoRI + NcoI, EB = EcoRI + BamHI]]
 +
[[File:DTU-Generuler.png|240px|thumb|right| The ladder used as supplied by [www.fermentas.com/templates/files/tiny_mce/coa_pdf/coa_sm0331.pdf Fermentas]]]
-
[[File:DTU-Strains_uninduced.GFPvsBiomass_v4.png|665px|thumb|center|add some text]]
+
The PCR to test the P<sub>tet</sub>-chiX fusion was done on a number of colonies following a transformation. The primers used were CS149 (GGTTTGCCACCTGACGTCTAAGAA) and CS150 (CCAAATTACCGCCTTTGAGTG). These two primers anneal just outside the BioBrick and can be used to PCR any BioBrick in a BioBrick compatible plasmid. The expected size of the PCR product was 1358 bp. Two of the colonies tested contained bands of the correct size, while the rest were background. These two were purified and subsequently used.
-
[[Image:DTU-Strains_induced.GFPvsBiomass_v4.png|665px|thumb|center|add some text]]
+
 
 +
[[File:DTU-ChiX.png|400px|thumb|left| Colony PCR of cells transformed with P<sub>tet</sub>-chiX inside pSB1C3, CS149 and CS150 primers were used. Expected band size is 1391 bp]]
 +
A number of P<sub>chiP</sub>-lacZ fusions were verified using restriction analysis. The expected band sizes are shown below, as is a gel-picture of the restriction:
 +
 
 +
{| class="wikitable" | center
 +
|+ P<sub>chiP</sub>-lacZ
 +
|-
 +
! Restriction Enzymes
 +
! Expected sizes
 +
|-
 +
| EcoRI + EcoRV + PstI
 +
| 1743 + 2101 + 1136 + 898
 +
|}
 +
<div style="clear: both;"></div>
 +
[[File:DTU1_ChiP_verification.png|665px|thumb|center| A linearized version of the P<sub>chiP</sub>-lacZ construct inside the pSB1C3 plasmid. Restriction sites used, as well as expected fragment sizes are indicated.]]
 +
 
 +
[[File:DTU-chip-lacZ.png|300px|thumb|right| A linearized version of the P<sub>chiP</sub>-lacZ construct inside the pSB1C3 plasmid. Restriction sites used, as well as expected fragment sizes are indicated.]]
 +
 
 +
==== Results and Conclusions ====
 +
Although we believe we obtained the correct plasmids, and they were subcloned into plasmids that let us test them, we were unable to do so due to time pressure. Only a few combinations with the P<sub>chiP</sub> plasmids were performed. These are summarized in the table below:
 +
 
 +
{| class="wikitable"
 +
|-
 +
! Strain
 +
! [http://ecoliwiki.net/colipedia/index.php/NM522 NM522]
 +
! [https://2011.igem.org/Team:DTU-Denmark/Project_testing_sRNA#Strain_construction IG302]
 +
|-
 +
| Genotype
 +
| chiX +
 +
| chiX -
 +
|-
 +
| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K564012 '''PchiP(original):lacZ, BBa_K564012''']
 +
| 467
 +
| 4781
 +
|-
 +
| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K564013 '''PchiP(mutated):lacZ, BBa_K564013''']
 +
| 2353
 +
| 2403
 +
|}
 +
 
 +
We concluded that the P<sub>chiP</sub> is regulated by chiX, and that mutating the Shine-Dalgarno (SD) removes the regulatory effect of chiX on the P<sub>chiP</sub> SD. The lower baseline expression of the mutated promoter is most likely due to the slightly weaker SD.
-
[[Image:DTU-SPL_uninduced.GFPvsBiomass_v4.png|665px|thumb|center|add some text]]
 
-
[[Image:DTU-SPL_induced.GFPvsBiomass_v4.png|665px|thumb|center|add some text]]
 
-
[[Image:DTU-Relative_promoter_activity.png|665px|thumb|center|add some text]]
 
<html></div><div class="whitebox article"></html>
<html></div><div class="whitebox article"></html>
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[5] Overgaard, Martin, Jesper Johansen, Jakob Møller‐Jensen, and Poul Valentin‐Hansen. “Switching off small RNA regulation with trap‐mRNA.” Molecular Microbiology 73, no. 5 (September 2009): 790-800.  
[5] Overgaard, Martin, Jesper Johansen, Jakob Møller‐Jensen, and Poul Valentin‐Hansen. “Switching off small RNA regulation with trap‐mRNA.” Molecular Microbiology 73, no. 5 (September 2009): 790-800.  
-
[6] Novick, A. & Weiner, M. “Enzyme induction as an all-or-none phenomenon“ Proc. Natl. Acad. Sci. USA 43 (1957), 533–556.
+
[6] Rasmussen, Anders Aamann, Jesper Johansen, Jesper S Nielsen, Martin Overgaard, Birgitte Kallipolitis, and Poul Valentin‐Hansen. “A conserved small RNA promotes silencing of the outer membrane protein YbfM.” Molecular Microbiology 72, no. 3 (May 2009): 566-577.
-
 
+
-
[7] Khlebnikov, A., K. Datsenko, T. Skaug, B. L. Wanner, and J. D. Keasling. “Homogeneous expression of the P(BAD) promoter in Escherichia coli by constitutive expression of the low-affinity high-capacity AraE transporter.” Microbiology (Reading, England) 147, no. Pt 12 (December 2001): 3241-7. http://www.ncbi.nlm.nih.gov/pubmed/11739756.
+
-
 
+
-
[8] Lee, Sung Kuk, and Jay D Keasling. “A Propionate-Inducible Expression System for Enteric Bacteria.” Society 71, no. 11 (2005): 6856-6862.
+
-
 
+
-
[9] Lee, Sung Kuk, and Jay D Keasling. “Propionate-Regulated High-Yield Protein Production in Escherichia coli.” Biotechnology (2005).
+
-
 
+
-
[10] Guzman, L M, D Belin, M J Carson, and J Beckwith. “Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter.” Journal of bacteriology 177, no. 14 (July 1995): 4121-30. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=177145&tool=pmcentrez&rendertype=abstract.
+
-
 
+
-
[11] Morgan-Kiss, Rachael M, Caryn Wadler, and John E Cronan. “Long-term and homogeneous regulation of the Escherichia coli araBAD promoter by use of a lactose transporter of relaxed specificity.” Proceedings of the National Academy of Sciences of the United States of America 99, no. 11 (May 28, 2002): 7373-7. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=124238&tool=pmcentrez&rendertype=abstract.
+
-
 
+
{{:Team:DTU-Denmark/Templates/Standard_page_end}}
{{:Team:DTU-Denmark/Templates/Standard_page_end}}

Latest revision as of 04:11, 22 September 2011

Experiment: Testing sRNA

Contents

Motivation

The biological experiments of the testing sRNA part is a proof of concept, showing that the E. coli sRNA sroB and the intergenic sRNA (trap-RNA) in the chbBCARG gene can be used to control gene expression by targeting the ybfM Shine-Dalgarno, and that these sRNAs can be rationally designed to target other Shine-Dalgarno sequences. The purpose is two-fold to construct plasmids and strains with deletions.

Strain construction involves deleting the original genes from the chromosome of a E. coli W3110 strain. Since we use the original genes, these need to be deleted from the chromosome to prevent them from interfering with our measurements.

Construction of plasmids necessary for testing our system involves taking the native system from E. coli, as well as a slightly modified system and putting them into plasmids that let us both control the expression of these components and measure the output of the system.

Strain construction

The strains used in testing our system were: IG9 a E. coli based on the wild-type K-12[3] strain W3110 with the lacZYA, chbBCARG operons deleted, and IG302 which is the K-12 $\Delta$lacZYA, $\Delta$chiP (alias ybfM), $\Delta$chiX (alias sroB, micM) strain constructed by Rasmussen et al.[6].

chbBCARG, chiP and chiX code for utilization of chitobiose (a sugar), chitobiose permease and the regulation of the chitobiose permease respectively.

We used recombineering[1] to delete these genes. Schematically the procedure works in the following manner:

  1. Transform competent cells with Red and Cre helper plasmids.
  2. Make the cells competent and induce the Red recombination system.
  3. Transform with linear piece of DNA.
  4. Select for gain of resistance.
  5. Do colony PCR to check that resistance gene is inserted in the correct place.
  6. Move to medium inducing the Cre recombinase.
  7. Screen for loss of resistance.
  8. Verify the construct by colony PCR.
  9. Repeat step 2-7 as needed.
  10. Cure the helper plasmids.

The Red helper plasmids encodes the $\lambda{}$ phage Red recombination system. This system allows recombination events between sequences with around 40 nucleotides of homology. The cre helper plasmid encodes the Cre recombinase. This recombinase recognises 34bp loxP sequences and promotes recombination between them.

An example of a linear piece of DNA that could be used with this method, as well as the simplified representation of the procedure, can be seen in figure:

A schematic overview of the Recombineering procedure. A linear piece of DNA, carrying a resistance marker is transformed into a strain carrying two helper plasmids, the $\lambda{}$ phage recombination system mediates integration of the linear piece of DNA. The Cre recombinase mediates the excision of the resistance marker.
The Cre recombinase recognizes the lox-sites. The mutated sites lox-71 and lox-66 are recognized, but the resulting lox-site is not.

In case of two or more subsequent gene deletions using Red-Cre recombineering a problem of recombination between an old loxP site and newly introduced ones arises. Remember, that after each round of redswap one loxP site is left in the genome. As a result recombination using Cre recombinase in second or next redswap might not occur between loxP sites flanking a resistance gene. To circumvent this problem two mutated loxP sequences are used - lox66 and lox71[4]. In spite of the mutations these lox sites are recognized by Cre which can remove the intervening region. Finally, from lox66 and lox71 a new site is formed, lox72, which has a reduced affinity to Cre recombinase. Thus, more than one gene deletions can be subsequently conducted.

Construction of plasmids

In order to quantitatively describe the natural trap-RNA system the following four plasmids were constructed:

  • PchiP-lacZ plasmid
To mimic and test repression of the chiP gene its upstream region of 599 bp was fused to reporter genes lacZ and inserted on the pSB1C3 plasmid. Expression from PchiP promoter is costitutive however it can be induced to a higher level by chitobiose. LacZ codes for $\beta$-galactosidase and its activity was tested using $\beta$-Gal assays.
  • PchiP(mutated)-lacZ plasmid
This plasmid is similar to the PchiP-lacZ plasmid, except the Shine-Dalgarno has been changed. This is expected to abolish or limit the regulatory effect of chiX.
  • Ptet-chiX plasmid
A schematic representation of the constructed plasmids, and the backbones we placed them in.
ChiX which is 84 bp long and regulates the wild-type chiP Shine-Dalgarno[2][5], was inserted into pSB4K under the control of the inducible promoter Ptet. Expression from Ptet promoter was induced using anhydrotetracycline.
  • PBAD-chiXR plasmid
A 284 bp long introgenic region from chbBCARG operon known to regulate chiX[2][5] was inserted into pSB3T5 plasmid. Expression from that plasmid was controlled by regulatory element from ara operon with arabinose as inducer.

Details on how the plasmids were constructed, including information on primers used, can be found in lab notebook.

Verifying constructs

The plasmids we constructed were verified in two ways. The PBAD-chiXR fusion and PchiP-lacZ fusion were tested by restriction analysis of the plasmid. And the Ptet-chiX fusion was tested by PCR.

The restriction of three plasmids tested revealed fragments of the expected sizes. These can be seen in the table below. The gel picture is also available.


PBAD-chiXR
Restriction Enzymes Expected sizes
EcoRI + BamHI 1150 + 2550
EcoRI + NcoI 2930 + 770
A linearized version of the PBAD-chiXR construct inside the pSB1C3 plasmid. Restriction sites used, as well as expected fragment sizes are indicated.
The gel picture of the PBAD-chiXR construct. The restriction enzymes used, and the sizes are indicated on the picture. EN = EcoRI + NcoI, EB = EcoRI + BamHI
The ladder used as supplied by [www.fermentas.com/templates/files/tiny_mce/coa_pdf/coa_sm0331.pdf Fermentas]

The PCR to test the Ptet-chiX fusion was done on a number of colonies following a transformation. The primers used were CS149 (GGTTTGCCACCTGACGTCTAAGAA) and CS150 (CCAAATTACCGCCTTTGAGTG). These two primers anneal just outside the BioBrick and can be used to PCR any BioBrick in a BioBrick compatible plasmid. The expected size of the PCR product was 1358 bp. Two of the colonies tested contained bands of the correct size, while the rest were background. These two were purified and subsequently used.

Colony PCR of cells transformed with Ptet-chiX inside pSB1C3, CS149 and CS150 primers were used. Expected band size is 1391 bp

A number of PchiP-lacZ fusions were verified using restriction analysis. The expected band sizes are shown below, as is a gel-picture of the restriction:

PchiP-lacZ
Restriction Enzymes Expected sizes
EcoRI + EcoRV + PstI 1743 + 2101 + 1136 + 898
A linearized version of the PchiP-lacZ construct inside the pSB1C3 plasmid. Restriction sites used, as well as expected fragment sizes are indicated.
A linearized version of the PchiP-lacZ construct inside the pSB1C3 plasmid. Restriction sites used, as well as expected fragment sizes are indicated.

Results and Conclusions

Although we believe we obtained the correct plasmids, and they were subcloned into plasmids that let us test them, we were unable to do so due to time pressure. Only a few combinations with the PchiP plasmids were performed. These are summarized in the table below:

Strain [http://ecoliwiki.net/colipedia/index.php/NM522 NM522] IG302
Genotype chiX + chiX -
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K564012 PchiP(original):lacZ, BBa_K564012] 467 4781
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K564013 PchiP(mutated):lacZ, BBa_K564013] 2353 2403

We concluded that the PchiP is regulated by chiX, and that mutating the Shine-Dalgarno (SD) removes the regulatory effect of chiX on the PchiP SD. The lower baseline expression of the mutated promoter is most likely due to the slightly weaker SD.

References

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