Difference between revisions of "Team:Sheffield/Parts"
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<h2><center>Strong constitutive promoter</center></h2> | <h2><center>Strong constitutive promoter</center></h2> | ||
| − | <p>During our project we have chosen 2 biobrick promoters (J23100 and J23106) and modified them to be suitable for direct cloning. Such an approach allows for amplification of the modified promoter without the need to carry out more cloning steps and modifications. We designed our biobricks as being composed of a working medium or strong promoter followed by a ribosomal binding site designed for E. coli strains. With such a design, anyone using our biobrick will be able to clone their construct directly in front of a desired gene and express their protein. Our biobricks were cloned into pSB1C3 plasmids as shown below. Due to difficulties with cloning, only the strong promoter was submitted to the registry (BBa_K2016000)</p> | + | <p>During our project we have chosen 2 biobrick promoters (J23100 and J23106) and modified them to be suitable for direct cloning. Such an approach allows for amplification of the modified promoter without the need to carry out more cloning steps and modifications. We designed our biobricks as being composed of a working medium or strong promoter followed by a ribosomal binding site designed for E. coli strains. With such a design, anyone using our biobrick will be able to clone their construct directly in front of a desired gene and express their protein. Our biobricks were cloned into pSB1C3 plasmids as shown below. Due to difficulties with cloning, only the strong promoter was submitted to the registry (BBa_K2016000). </p> |
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<img style="width:40%;border:6px solid black" src="https://static.igem.org/mediawiki/2016/5/56/StrP.png"> | <img style="width:40%;border:6px solid black" src="https://static.igem.org/mediawiki/2016/5/56/StrP.png"> | ||
Revision as of 15:26, 18 October 2016
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BIOBRICKS |
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Introduction
Following our project we submitted 4 biobricks to iGEM 2016 registry. 2 of the biobricks included different hemerythrins genes: mc and td; one was an sfgfp gene under control of a strong promoter with an additional regulatory region operated by RyhB; the last biobricks was a modified strong promoter followed by a ribosomal binding site. Promoter sequences were modified using the iGEM biobrick Part:BBa_J23100. All submitted biobricks are summarised in the table below. Unfortunately we were unable to clone Dcr, medium promoter and Med-RyhB-GFP into our pSB1C3 plasmid, due to XbaI and SpeI restriction sites having the same sticky-ends, thus creating self-ligated plasmid on multiple occasions.
BBa K2016000 |
BBa K2016003 |
BBa K2016004 |
BBa K2016005 |
Strong constitutive promoter
During our project we have chosen 2 biobrick promoters (J23100 and J23106) and modified them to be suitable for direct cloning. Such an approach allows for amplification of the modified promoter without the need to carry out more cloning steps and modifications. We designed our biobricks as being composed of a working medium or strong promoter followed by a ribosomal binding site designed for E. coli strains. With such a design, anyone using our biobrick will be able to clone their construct directly in front of a desired gene and express their protein. Our biobricks were cloned into pSB1C3 plasmids as shown below. Due to difficulties with cloning, only the strong promoter was submitted to the registry (BBa_K2016000).
Figure 1: Map of BBa_K2016000 biobrick cloned into pSC1C3 plasmid. Biobrick is cloned between XbaI and SpeI sites
McHr - hemerythrin
Methylococcus capsulatus hemerythrin (McHr) is a protein expressed by methane-oxidising bacterium: Methylococcus capsulatus. The family of hemerythrins can undergo a slow process of oxygenation upon binding of iron which causes their colour change from colourless into red/yellow. Its mechanism of oxygenation has been discovered to be much more rapid than other hemerythrins we investigated (DcrHr or TdHr). This gene was synthesised based on the GeneBank sequence entry and cloned into the pSB1C3 plasmid.
Figure 1: Map of BBa_K2016003 biobrick cloned into pSC1C3 plasmid. Biobrick is cloned between XbaI and SpeI sites, however it is cloned in reverse orientation due to XbaI and SpeI enzymes having the same overhangs after restriction digest. To use this biobrick future teams should use PCR apmlification rather than restriction digest to clone this biobrick.
TdHr - hemerythrin
TdHr is a protein expressed by Themiste dyscritum, a eukaryotic marine organism, in which this protein functions as an iron-binding transporter, much like our own haemoglobin. Td undergoes a slow process of oxygenation upon binding of iron and gains a violet/pink colour. This gene was codon-optimised for the use in E. coli and cloned into the pSB1C3 plasmid.
Figure 1: Map of BBa_K2016004 biobrick cloned into pSC1C3 plasmid. Biobrick is cloned between XbaI and SpeI sites.
Str-RyhB-GFP - Superfolder GFP under the control of RyhB
Our superfolder GFP was cloned to be operated by RyhB on the RNA interference level. RyhB is responsive to iron concentrations within the cell and therefore provides us with a viable tool needed for iron detection. Strong or medium promoters were used for expression of gfp on different levels. However, due to difficulties with cloning, only the GFP under control of strong promoter was submitted to the iGEM registry. The gene was cloned into pSB1C3 vector as shown below.
Figure 1: Map of BBa_K2016005 biobrick cloned into pSC1C3 plasmid. Biobrick is cloned between XbaI and SpeI sites.
