Difference between revisions of "Part:BBa K4073000:Design"
 
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BBa_K407300 regulates salt tolerance in plants
 
  
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This composite part consists of a CrGDPH3 salt-inducible promoter, a medium-strength RBS, the AtHKT1 gene, and a terminator. The regulation of the expression of the AtHKT1 gene is the function of this part. The CrGPDH3 salt-inducible promoter was used for the overexpression of proteins in a transgene in microalga Chlamydia reinhardtii.  We then decided to utilize this promoter because of its high NaCl response elements. The RBS initiates translation of the HKT1 gene, which is essential for its protein synthesis. While we were unable to test the effects of the strength of the RBS in a lab due to COVID-19, if we were able to, we would have used ribosome profiling. That would have provided us with detection and a quantitative amount of translation in cells. Ribosome profiling requires first, a collection of a physiological sample which must freeze ribosomes while they’re in the midst of translation in order to produce isolated ribosome fragments from which ribosome footprints can be created and consequently sequenced to the appropriate genome. With the promoter and medium strength RBS, we would hope to see that translation is occurring with a high salt concentration. Once activated, the HKT1 gene should create the membrane protein AtKHT1, which is responsible for transporting sodium ions away from the sylem into xylem parenchyma cells. While we applied this part in order to remove sodium ions from the xylem of A. Thaliana, users can also use the HKT1 gene in other plant species with excess sodium ions. Finally, we used a composite terminator which signals the end of transcription in our part. This is essential, as if we did not utilize a terminator, the AtHKT1 gene could potentially be over-expressed, leading to decreased salt concentrations which could inhibit plant growth (salt is an essential nutrient used in plant growth). Lastly, if we had access to a lab, we would also consider gathering experimental data on the expression of the AtHKT1 and its effects on plants. We would gather data on plant growth to see if this part is beneficial for plants.
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<partinfo>BBa_K4073000 short</partinfo>
  
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The source of the medium strength RBS, HKT1 gene, and terminator all came from the iGEM registry. The RBS is the part labeled Bba_B0032, the HKT1 gene is the protein coding sequence of the part labeled "BBa_K2665007”, while the terminator is the part "BBa_B0015." The promoter was derived from the NCBI sequence for the CrGPDH3 salt-inducible promoter.
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<partinfo>BBa_K4073000 SequenceAndFeatures</partinfo>
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===Design Notes===
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Some design considerations that we had to deal with were the regions of the promoter that we wanted to focus on. For example, both RIA1 and RIA3 were regions of interest, and so we had to decide which one of them would yield the most enhanced salinity mechanisms in the form of NaCl response elements. We ended up choosing the restriction enzymes SpeI to KpnI, encompassing both the RIA1 and the RIA3 regions, for the maximum effect of the mechanisms under theoretical NaCl treatments. Additionally, a medium strength RBS was used so that a decent strength of expression of the HKT1 gene was achieved (our other composite parts touch upon the other RBS strengths). Another important aspect to consider is that our design could have had the capability to be altered based on how experimental lab procedures followed through. However, due to the lack of access to a lab, we were unable to test the effects of this composite part on the salinity mechanisms of Arabidopsis Thaliana, and therefore most of our design considerations were centered around research gathered from original papers (https://link.springer.com/content/pdf/10.1007/s00253-019-09733-y.pdf).
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===Source===
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The source of the medium strength RBS, HKT1 gene, and terminator all came from the iGEM registry. The RBS is the part labeled Bba_B0032, the HKT1 gene is the protein coding sequence of the part labeled "BBa_K2665007”, while the terminator is the part "BBa_B0015." The promoter was derived from the NCBI sequence (​​https://www.ncbi.nlm.nih.gov/nuccore/1601834299) for the CrGPDH3 salt-inducible promoter.  
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===References===

Latest revision as of 06:43, 17 October 2021


BBa_K4073000 regulates salt tolerance in plants.


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 755
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1912
    Illegal BsaI.rc site found at 225


Design Notes

Some design considerations that we had to deal with were the regions of the promoter that we wanted to focus on. For example, both RIA1 and RIA3 were regions of interest, and so we had to decide which one of them would yield the most enhanced salinity mechanisms in the form of NaCl response elements. We ended up choosing the restriction enzymes SpeI to KpnI, encompassing both the RIA1 and the RIA3 regions, for the maximum effect of the mechanisms under theoretical NaCl treatments. Additionally, a medium strength RBS was used so that a decent strength of expression of the HKT1 gene was achieved (our other composite parts touch upon the other RBS strengths). Another important aspect to consider is that our design could have had the capability to be altered based on how experimental lab procedures followed through. However, due to the lack of access to a lab, we were unable to test the effects of this composite part on the salinity mechanisms of Arabidopsis Thaliana, and therefore most of our design considerations were centered around research gathered from original papers (https://link.springer.com/content/pdf/10.1007/s00253-019-09733-y.pdf).


Source

The source of the medium strength RBS, HKT1 gene, and terminator all came from the iGEM registry. The RBS is the part labeled Bba_B0032, the HKT1 gene is the protein coding sequence of the part labeled "BBa_K2665007”, while the terminator is the part "BBa_B0015." The promoter was derived from the NCBI sequence (​​https://www.ncbi.nlm.nih.gov/nuccore/1601834299) for the CrGPDH3 salt-inducible promoter.

References