Difference between revisions of "Team:Birkbeck/Basic Parts"
| Line 18: | Line 18: | ||
<img src="https://static.igem.org/mediawiki/2015/f/fd/Birkbeck_stf_phage.png"> | <img src="https://static.igem.org/mediawiki/2015/f/fd/Birkbeck_stf_phage.png"> | ||
<p>The <i>stf</i> gene encodes the short tail fibres found in the Ur-lambda bacteriophage, the original isolate [1]. The wild type lambda strain (λ-PaPa) that is most commonly used in research carries a frameshift mutation in the <i>stf</i> gene sequence producing no such fibres. It has been shown that the presence of these additional thin tails results in the more efficient adsorption to the host, <i>E.coli</i> [1]. | <p>The <i>stf</i> gene encodes the short tail fibres found in the Ur-lambda bacteriophage, the original isolate [1]. The wild type lambda strain (λ-PaPa) that is most commonly used in research carries a frameshift mutation in the <i>stf</i> gene sequence producing no such fibres. It has been shown that the presence of these additional thin tails results in the more efficient adsorption to the host, <i>E.coli</i> [1]. | ||
| + | <p></p> | ||
The <i>stf</i> gene is a product of two open reading frames: ORF-401 and ORF-314. Introduction of 1 bp just after ORF-401 removes the frameshift fusing the two coding regions together which results in a functional short tail fibre protein consisting of 774 aa [2]. The C-terminal end of the stf protein (i.e. ORF-314) offers host receptor recognition for OmpC (Outer membrane protein C) on the surface of <i>E.coli</i>, widening receptor specificity and host range relative to λ-PaPa which only recognises the <i>E.coli</i> Maltoporin (<i>lamB</i> gene product) through its tip attachment protein J [1],[3]. Additionally, the C-terminus (ORF-314) displays a high level of homology with the gp37 of bacteriophage T4 [4]. This BioBrick was registered as part <a href="http://parts.igem.org/Part:BBa_K1846004">BBa_K1846004</a>.</p> | The <i>stf</i> gene is a product of two open reading frames: ORF-401 and ORF-314. Introduction of 1 bp just after ORF-401 removes the frameshift fusing the two coding regions together which results in a functional short tail fibre protein consisting of 774 aa [2]. The C-terminal end of the stf protein (i.e. ORF-314) offers host receptor recognition for OmpC (Outer membrane protein C) on the surface of <i>E.coli</i>, widening receptor specificity and host range relative to λ-PaPa which only recognises the <i>E.coli</i> Maltoporin (<i>lamB</i> gene product) through its tip attachment protein J [1],[3]. Additionally, the C-terminus (ORF-314) displays a high level of homology with the gp37 of bacteriophage T4 [4]. This BioBrick was registered as part <a href="http://parts.igem.org/Part:BBa_K1846004">BBa_K1846004</a>.</p> | ||
Revision as of 17:02, 16 November 2015
Our BioBricks
Basic Parts
ORF314 (BBa_K1846000)
In the commonly used lab strain of λ-bacteriophage (known as λPaPa), ORF314 is one of two open reading frames resulting from a frameshift in the tail fibre protein gene (stf). This ORF codes for the C-terminus of the protein, which contains the host receptor recognition site. ORF314 binds to the OmpC protein of E.coli. Furthermore, the sequence shows great (>50%) homology with the gene 37 tail fibre protein of bacteriophage T4.
To transform this sequence into a BioBrick basic part, we had the coding sequence synthesised, including the BioBrick prefix and suffix and an additional section to facilitate the cloning of complementary sequence ORF401. We then restricted both our synthesised gene and the linearised plasmid backbones (pSB1C3 for shipping and pSB1K3 for further processing) with EcoRI and PstI, before ligating the two pieces together using T4 DNA ligase. Success of the cloning procedure was confirmed by restriction with EcoRI and PstI followed by DNA agarose electrophoresis (Fig. 1), with pSB1C3 alone and ORF314 alone as controls. The ORF314 biobrick has been assigned BBa_K1846000.
Fig 1. Agarose gel electrophoresis of ORF314 inserted in pSB1C3 and restricted with EcoRI and PstI. The expected band sizes are 1 kb for ORF314 and 2 kb for pSB1C3. Successful results were obtained for samples 2 and 3.
stf (short tail fibre) gene (BBa_K1846004)
The stf gene encodes the short tail fibres found in the Ur-lambda bacteriophage, the original isolate [1]. The wild type lambda strain (λ-PaPa) that is most commonly used in research carries a frameshift mutation in the stf gene sequence producing no such fibres. It has been shown that the presence of these additional thin tails results in the more efficient adsorption to the host, E.coli [1].
The stf gene is a product of two open reading frames: ORF-401 and ORF-314. Introduction of 1 bp just after ORF-401 removes the frameshift fusing the two coding regions together which results in a functional short tail fibre protein consisting of 774 aa [2]. The C-terminal end of the stf protein (i.e. ORF-314) offers host receptor recognition for OmpC (Outer membrane protein C) on the surface of E.coli, widening receptor specificity and host range relative to λ-PaPa which only recognises the E.coli Maltoporin (lamB gene product) through its tip attachment protein J [1],[3]. Additionally, the C-terminus (ORF-314) displays a high level of homology with the gp37 of bacteriophage T4 [4]. This BioBrick was registered as part BBa_K1846004.tfa gene (BBa_K1846002)
Having the tail fibre assembly gene of bacteriophage lambda synthesised as a construct including a promoter, RBS and terminator, and cloned the entire construct into the shipping backbone for submission (BioBrick BBa_K1846001), we then proceeded to use primers to remove the promoter, RBS and terminator from the sequence. We then cloned the gene alone into a vector for submission as a separate basic part. This BioBrick was registered as part BBa_K1846002. Unfortunately, sequencing and agarose gel electrophoresis suggested we had obtained an incorrect product, and as such the part has not yet been submitted as a BioBrick.
J gene (BBa_K1846008)
The J gene of bacteriophage lambda encodes the tip attachment protein that forms the base plate of the bacteriophage's tail. It is responsible for host recognition and binding, forming bonds to the lamB porin found in the E.coli outer membrane. We obtained this sequence as part of a collection of bacteriophage lambda parts from Technion, Haifa, Israel; this specific sequence was part of their previous BioBrick BBa_K784017. Having initially created primers to excise the gene from the surrounding sequence, we then attempted to use Gibson assembly to remove several illegal PstI restriction sites. Unfortunately, the length of the various sequences caused this experiment to be usuccessful. We are currently still attempting to remove two of these restriction sites. The BioBrick has been registered as part BBa_K1846008. However, due to difficulties removing the illegal restriction sites the part has not as yet been submitted to the registry.