Difference between revisions of "Part:BBa K3745030:Design"

(→‎Producing and Quantifying Rhamnolipid)
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===Producing and Quantifying Rhamnolipid===
 
===Producing and Quantifying Rhamnolipid===
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Our team aims to produce rhamnolipid by using BBa_K2745030 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 futher propel the production of rhamnolipids by measure the amount of production of rhamnolipids by using the HPLC-MS method. The specific quantification steps are shown below.
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Our team aims to produce rhamnolipid by using BBa_K2745030 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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Quantification using HPLC-MS
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1. Add HCl into the liquid culture until the final pH is 2
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2. Leave the solution overnight at 4° C
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3. Extract the organic phase using an ethanol-chloroform solution in a ratio of 1:2.
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4. HPLC-MS is performed using C18 column and acetonitrile as mobile phase.
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Revision as of 14:21, 11 October 2021


Plac+rhlABC linear fragment


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 3524
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 2185
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 2149
    Illegal NgoMIV site found at 2458
    Illegal NgoMIV site found at 4067
    Illegal AgeI site found at 3070
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 4206


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

Producing and Quantifying Rhamnolipid

Our team aims to produce rhamnolipid by using BBa_K2745030 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.