Difference between revisions of "Part:BBa K2127001:Design"

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===Design Notes===
 
===Design Notes===
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Cyanobacteria is an not optimal system to manipulate the protein expression mainly due of the lack of strong promoters. Pcpc560 is a Cyanobacterial super promoter discovered by Dr. Ma’s team that contains two predicted promoter sites and a transcription binding site containing 14 homologous bacterial regions upstream of the regulated cpcB gene. Using Pcpc560, Dr. Ma’s team demonstrated that functional protein was produced at 15% of the total cell protein volume. Our team decided to test the effect of the transcription factor binding site on protein production in E.Coli cells DH5α. We believe that the predicted 14 homologous binding sites from Pcpc560 will allow us to increase the protein production greatly using the E.Coli DH5α. We constructed a biobrick containing the 14 transcription binding sites followed by a strong RBS (BBa_B0030) and a mutant HIS-Tag psbB gene. The super promoter can be an valuable asset to other iGEM teams for protein production and therefore we have decided to submit the Construct along with 14 Transcription binding site from Pcpc560.
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BBa_K2127001 was designed to be expressible in E. coli to allow for easier characterization and study of the Transcription Factor Binding site (TFBS) obtained from Synechocystis PCC 6803. As shown by Dr MA with the study on promotor Cpc560, the TFBS showed a distinct effect on the ability to upregulate protein expression of a gene under promotor control. To see about developing a viable high expression and inducible promotor in for cyanobacterium, this TFBS was combined with a lac promotor under LacI regulation. The construct was then combined with our characterized His tagged psbB gene to determine and analyze the expression levels of the CP47His subunit. The construct was then combined with double stop codons and our own derivative of the BBa_B0015 double terminator (BBa_K2127007) to ensure that only the subunit will be expressed.
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To test the construct, E coli DH5alpha F' Iq was chosen as a suitable transformation and expression host.This is due to the ease of transformation into this host and the presence of the mutated strong lac repressor. The idea of the system is to facilitate the future work involving photosystem components within well characterized organisms such as E coli. Consequentially, we also aim to develop a way to create inducible systems within cyanobacteria that will allow for easier genetic and protein work.
  
 
===Source===
 
===Source===

Revision as of 14:29, 21 October 2016


Inducible High Expression His-Tagged Photosystem II CP47 subunit


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 658
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 782
    Illegal AgeI site found at 1379
    Illegal AgeI site found at 1583
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1103
    Illegal SapI site found at 1780


Design Notes

BBa_K2127001 was designed to be expressible in E. coli to allow for easier characterization and study of the Transcription Factor Binding site (TFBS) obtained from Synechocystis PCC 6803. As shown by Dr MA with the study on promotor Cpc560, the TFBS showed a distinct effect on the ability to upregulate protein expression of a gene under promotor control. To see about developing a viable high expression and inducible promotor in for cyanobacterium, this TFBS was combined with a lac promotor under LacI regulation. The construct was then combined with our characterized His tagged psbB gene to determine and analyze the expression levels of the CP47His subunit. The construct was then combined with double stop codons and our own derivative of the BBa_B0015 double terminator (BBa_K2127007) to ensure that only the subunit will be expressed.

To test the construct, E coli DH5alpha F' Iq was chosen as a suitable transformation and expression host.This is due to the ease of transformation into this host and the presence of the mutated strong lac repressor. The idea of the system is to facilitate the future work involving photosystem components within well characterized organisms such as E coli. Consequentially, we also aim to develop a way to create inducible systems within cyanobacteria that will allow for easier genetic and protein work.

Source

Synchocisytys

References