Difference between revisions of "Part:BBa K3745043:Design"

 
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===Design Notes===
 
===Design Notes===
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na
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It is worth informing that the Plac in the DNA feature's graph is a reverse promoter (activate LacI instead of activating rhlA, B, and C). The arrow on the feature is not accurate due to the reason that the image of the promoter won't appear in the reverse direction. Nevertheless, the DNA sequence is posted in the correct arrangement which Plac is responsible for activating LacI.
  
  
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===Source===
 
===Source===
  
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This study
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===Results===
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Our team aims to produce rhamnolipid by using BBa_K3745030 plasmid including Plac, rhlA, rhlB, and rhlC. First, IPTG is involved in our production process due to the reason that IPTG is a substance that is similar to lactose. In other words, it can be used to induce Plac and further propel the production of rhamnolipids by activating rhlA, rhlB, and rhlC. Therefore, our team also aims to carry out a quantification analysis of rhamnolipid production by using the HPLC-MS method. The specific quantification steps are shown below. To start with, we add HCl into the liquid culture until the final pH is 2. Then, the solution is left at 4° C overnight. After that, the organic phase is extracted using an ethanol-chloroform solution in a ratio of 1:2. Eventually, HPLC-MS is performed using C18 column and acetonitrile as mobile phase.
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Rhamnolipid is further tested using HPLC-MS. According to previous research (Liu et al, 2014), the high intensity at 503.0 and 677.7 Da is observed in pure rhamnolipid, at 503.0 is monorhamnolipid with C10-C12 tail and at 677.7 is dirhamnolipid with C10-C10 tail. Therefore, the production of rhamnolipid is successful. (please see HPLC-MS figure in the page of Engineering Success)
  
 
===References===
 
===References===
 +
1. LIU, Y., ZHONG, H., LIU, Z., JIANG, Y., TAN, F., ZENG, G., LAI, M., & HE, Y. (2014). Purification and characterization of the biosurfactant rhamnolipid. Chinese Journal of Chromatography, 32(3), 248. https://doi.org/10.3724/sp.j.1123.2013.10026
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2. Han, L., Liu, P., Peng, Y., Lin, J., Wang, Q., & Ma, Y. (2014). Engineering the biosynthesis of novel rhamnolipids in Escherichia coli for enhanced oil recovery. Journal of Applied Microbiology, 117(1), 139–150. https://doi.org/10.1111/jam.12515 ‌ ‌

Latest revision as of 08:08, 21 October 2021


Plac+rhlABC


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 7
    Illegal NotI site found at 3546
    Illegal NotI site found at 4759
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 2171
    Illegal NgoMIV site found at 2480
    Illegal NgoMIV site found at 4089
    Illegal AgeI site found at 3092
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 4228


Design Notes

It is worth informing that the Plac in the DNA feature's graph is a reverse promoter (activate LacI instead of activating rhlA, B, and C). The arrow on the feature is not accurate due to the reason that the image of the promoter won't appear in the reverse direction. Nevertheless, the DNA sequence is posted in the correct arrangement which Plac is responsible for activating LacI.


Source

This study

Results

Our team aims to produce rhamnolipid by using BBa_K3745030 plasmid including Plac, rhlA, rhlB, and rhlC. First, IPTG is involved in our production process due to the reason that IPTG is a substance that is similar to lactose. In other words, it can be used to induce Plac and further propel the production of rhamnolipids by activating rhlA, rhlB, and rhlC. Therefore, our team also aims to carry out a quantification analysis of rhamnolipid production by using the HPLC-MS method. The specific quantification steps are shown below. To start with, we add HCl into the liquid culture until the final pH is 2. Then, the solution is left at 4° C overnight. After that, the organic phase is extracted using an ethanol-chloroform solution in a ratio of 1:2. Eventually, HPLC-MS is performed using C18 column and acetonitrile as mobile phase.

Rhamnolipid is further tested using HPLC-MS. According to previous research (Liu et al, 2014), the high intensity at 503.0 and 677.7 Da is observed in pure rhamnolipid, at 503.0 is monorhamnolipid with C10-C12 tail and at 677.7 is dirhamnolipid with C10-C10 tail. Therefore, the production of rhamnolipid is successful. (please see HPLC-MS figure in the page of Engineering Success)

References

1. LIU, Y., ZHONG, H., LIU, Z., JIANG, Y., TAN, F., ZENG, G., LAI, M., & HE, Y. (2014). Purification and characterization of the biosurfactant rhamnolipid. Chinese Journal of Chromatography, 32(3), 248. https://doi.org/10.3724/sp.j.1123.2013.10026

2. Han, L., Liu, P., Peng, Y., Lin, J., Wang, Q., & Ma, Y. (2014). Engineering the biosynthesis of novel rhamnolipids in Escherichia coli for enhanced oil recovery. Journal of Applied Microbiology, 117(1), 139–150. https://doi.org/10.1111/jam.12515 ‌ ‌