IGEM:IMPERIAL/2008/Prototype/Wetlab/parts

Adding Parts to the Registry

The registry has a simple guide about adding parts on the following link. Before we start to add our parts we should collect the following information about each of our parts:

• A part name
• The DNA sequence of the part you are making
• A short description of the part
• A long description of the part, including references
• The source of the part, including references
• Design Considerations

Most of this information is on the wiki under the [Sequence Page]. If the wet lab team could all contribute to adding these parts it would help speed things up. Once we have our parts we can then build up our constructs that we will be submitting to the registry.

Promoters

Promoter ctc

• Part name = promoter ctc
• Sequence =
• Promoter ctc is a sigma factor B dependent promoter found in B.subtilis. In B.subtilis endogenous sigma factor B is activated under mild stress from nutrient and physical stress response. The context with which we used the promoter ctc, was to take blue light as an input and give Polymerase Per Second(PoPS) as an output.

• Promoter ctc is a sigma factor B dependent promoter found in B.subtilis. In B.subtilis endogenous sigma factor B is activated under mild stress. These mild stress conditions can be generally split into nutrient stress response and physical stress response. Nutrient stress response is triggered by low levels of ATP and GTP and physical stress response is triggered by exposure to blue light, salt, heat, acid or ethanol[1]. The promoter ctc has been used previously as a read out for the activation of sigma factor B [2].
• The context with which we used the promoter ctc, was to take blue light as an input and give Polymerase Per Second(PoPS) as an output. To do this the other potential inputs need to be carefully controlled so that only blue light activated the sigma B and gives a PoPS output. In order to get sufficient sigma B activation by blue light the light receptor YtvA, part...., needs to be over expressed in B.subtilis [3].

• Source - The part was designed using the sequence from the B.subtilis genome and from previously published papers [2][3]. This sequence was then synthesised by Geneart.

• Design - Biobrick standard was applied to the promoter ctc sequence.

References

1. Zhang S and Haldenwang WG. Contributions of ATP, GTP, and redox state to nutritional stress activation of the Bacillus subtilis sigmaB transcription factor. J Bacteriol. 2005 Nov;187(22):7554-60. DOI:10.1128/JB.187.22.7554-7560.2005 |
2. Igo MM and Losick R. Regulation of a promoter that is utilized by minor forms of RNA polymerase holoenzyme in Bacillus subtilis. J Mol Biol. 1986 Oct 20;191(4):615-24. DOI:10.1016/0022-2836(86)90449-3 |
3. Suzuki N, Takaya N, Hoshino T, and Nakamura A. Enhancement of a sigma(B)-dependent stress response in Bacillus subtilis by light via YtvA photoreceptor. J Gen Appl Microbiol. 2007 Apr;53(2):81-8. DOI:10.2323/jgam.53.81 |

Promoter gsiB

• Part name = promoter gsiB
• Sequence =
• Promoter gsiB is a sigma factor B dependent promoter found in B.subtilis. In B.subtilis endogenous sigma factor B is activated under mild stress from nutrient and physical stress response. The context with which we used the promoter gsiB, was to take blue light as an input and give Polymerase Per Second(PoPS) as an output.

• Promoter gsiB is a sigma factor B dependent promoter found in B.subtilis. In B.subtilis endogenous sigma factor B is activated under mild stress. These mild stress conditions can be generally split into nutrient stress response and physical stress response. Nutrient stress response is triggered by low levels of ATP and GTP and physical stress response is triggered by exposure to blue light, salt, heat, acid or ethanol[1]. The promoter gsiB has been used previously as a read out for the activation of sigma factor B [2].
• The context with which we used the promoter gsiB, was to take blue light as an input and give Polymerase Per Second(PoPS) as an output. To do this the other potential inputs need to be carefully controlled so that only blue light activated the sigma B and gives a PoPS output. In order to get sufficient sigma B activation by blue light the light receptor YtvA, part...., needs to be over expressed in B.subtilis [3].

• Source - The part was designed using the sequence from the B.subtilis genome and from previously published papers [2][3]. This sequence was then synthesised by Geneart.

• Design - Biobrick standard was applied to the promoter gsiB sequence.

References

1. Zhang S and Haldenwang WG. Contributions of ATP, GTP, and redox state to nutritional stress activation of the Bacillus subtilis sigmaB transcription factor. J Bacteriol. 2005 Nov;187(22):7554-60. DOI:10.1128/JB.187.22.7554-7560.2005 |
2. Nguyen HD, Nguyen QA, Ferreira RC, Ferreira LC, Tran LT, and Schumann W. Construction of plasmid-based expression vectors for Bacillus subtilis exhibiting full structural stability. Plasmid. 2005 Nov;54(3):241-8. DOI:10.1016/j.plasmid.2005.05.001 |
3. Suzuki N, Takaya N, Hoshino T, and Nakamura A. Enhancement of a sigma(B)-dependent stress response in Bacillus subtilis by light via YtvA photoreceptor. J Gen Appl Microbiol. 2007 Apr;53(2):81-8. DOI:10.2323/jgam.53.81 |

Integration Sequences

AmyE

5'

Name: 5’ AmyE Integration Sequence

Code: BBa_K143001

Sequence:

Short: 5’ integration sequence for the AmyE locus of B.subtilis

Long: The 5' integration sequence can be added to the front of a Biobrick construct and the 3' integration sequence specific for this locus (Part BBa_K143002) to the rear of the Biobrick construct to allow integration of the Biobrick construct into the chromosome of the gram positive bacterium B.subtilis. The 5' and 3' integration sequences for the AmyE locus were used to integrate the Imperial 2008 iGEM project primary construct into the B.sutbilis chromosome.

Source: The 5’ sntegration sequence was taken from the shuttle vector pDR111 which has been used

Design: The AmyE integration sequence was taken from the vector after comparison by BLAST to the B.subtilis chromosome to identify the homologous sequences. The sequence present in both the host chromosome and the plasmid at the 5' end of the gene is the 5' sequence required for integration