Difference between revisions of "Part:BBa K3031016"
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This system was used to test the low iron environment.  
 
This system was used to test the low iron environment.  
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<h3>Experiment</h3>
 
<h3>Experiment</h3>
 
<p>To test the effectiveness of our new part luxI promoter with FUR - we needed to expose cells containing transformed plasmid into both iron rich and iron starved environments. Single colonies were inoculated in 50 ml LB broth containing Ampicillin in a 1000:1 ratio and 40 μM FeSO4 in Falcon tubes and cultured at 37 C until OD600 = 0.5. 10ml culture was added to each of three 15ml tubes. Sample A contains blank cell (without plasmid) culture. Sample B contains culture (with plasmid) with 200 μM DP (2,2'-Dipyridine). The function of the 2,2'-Dipyridine is to remove iron in the cellular environment and thus mimic the low iron environment of the gut. Sample C contains only the culture (with plasmid) without any 2,2'-Dipyridine.</p>
 
<p>To test the effectiveness of our new part luxI promoter with FUR - we needed to expose cells containing transformed plasmid into both iron rich and iron starved environments. Single colonies were inoculated in 50 ml LB broth containing Ampicillin in a 1000:1 ratio and 40 μM FeSO4 in Falcon tubes and cultured at 37 C until OD600 = 0.5. 10ml culture was added to each of three 15ml tubes. Sample A contains blank cell (without plasmid) culture. Sample B contains culture (with plasmid) with 200 μM DP (2,2'-Dipyridine). The function of the 2,2'-Dipyridine is to remove iron in the cellular environment and thus mimic the low iron environment of the gut. Sample C contains only the culture (with plasmid) without any 2,2'-Dipyridine.</p>
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<h3>Usage and Applications</h3>
 
<h3>Usage and Applications</h3>
  

Revision as of 11:56, 21 October 2019


Iron QS system + GFP reporter

This system is designed to express downstream genes of interest only in low iron environments. The LuxI/LuxR QS system ensures only expression of proteins once a high cell density is reached. We have used the modified LuxI promoter designed by this team to regulate expression of downstream genes using a Ferric Uptake Regulator repressor. The QS system is now inhibited as long as ferric iron is available to the cell as LuxI protein is not produced, resulting in no AHL + LuxR complex forming. Therefore the Lux promoter (pLux) is not activated.

Activation in the system is achieved in low iron environments such as the guts of fish. This makes the system ideal for an oral vaccine delivery system where antigen production could be controlled until the cell reaches the gut of the fish after ingestion.

This system was used to test the low iron environment.

Experiment

To test the effectiveness of our new part luxI promoter with FUR - we needed to expose cells containing transformed plasmid into both iron rich and iron starved environments. Single colonies were inoculated in 50 ml LB broth containing Ampicillin in a 1000:1 ratio and 40 μM FeSO4 in Falcon tubes and cultured at 37 C until OD600 = 0.5. 10ml culture was added to each of three 15ml tubes. Sample A contains blank cell (without plasmid) culture. Sample B contains culture (with plasmid) with 200 μM DP (2,2'-Dipyridine). The function of the 2,2'-Dipyridine is to remove iron in the cellular environment and thus mimic the low iron environment of the gut. Sample C contains only the culture (with plasmid) without any 2,2'-Dipyridine.


After induction with DP for 4 hours, 1 ml of each cell culture broth was transferred to two 1.5 ml sterile centrifuge tubes and centrifuged at 4000rpm for 4 minutes. After removing the supernatant, we wash the cell with PBS buffer. Then, 100 μM culture was added into 96 well white polystyrene microplate and black polystyrene microplate, each with three samples. We measured the OD600 and Fluorescence (Excitation: 485nm/ Emission: 528nm) by using plate reader. The data was recorded. After that, we calculate the average OD600 and Fluorescence for each sample. For each of samples, we divided the relative fluorescence value (RFV) by the average OD600. This quantitative test was used to determine Fur and luxI/luxR-controlled protein expression under iron deprivation in E. coli.


  • Sample A = Blank (E.coliBL21(DE3) cells with no plasmid)
  • Sample B = E.coliBL21(DE3) cells containing our ironQS system (BBa_K3031016) and grown in iron rich media PLUS iron chelator 2,2'-Dipyridine
  • Sample C = E.coliBL21(DE3) cells containing our ironQS system (BBa_K3031016) and grown in iron rich media only.


IronQS Systems
Results of ironQS system testing. The graph shows for sample B, when our new luxI+FUR promoter was grown in the presence of 2,2'-Dipyridine, all the Fe2+ is released likely released from FUR and thus N-acylhomoserine lactone (AHL) molecules are produced which bind to LuxR molecules up-regulating the expression of GFP which is under control of the Lux promoter. The function of the 2,2'-Dipyridine is to remove iron in the cellular environment and thus mimic the low iron environment of the gut which allowed for the eventual expression of GFP. In sample C, when E.coli Bl21(DE3) cells were grown in the presence of iron (i.e. no 2,2'-Dipyridine) the ferric iron stays bound to the FUR box sequence and allows for the repression of the LuxR/LucI Quroum Sensing system. Sample A shows the blank control which contained no engineered plasmid and thus the fluorescence from sample A is attributed to normal cell metabolism. The fact that Samples A and C show similar results suggests the repression of the LuxR/luxI system was tightly controlled by ferric iron binding to the fur box and inhibiting the production of AHL. Our measurements show relative fluorescence values of BL21 cell containing ironQS system after cultured in iron-rich and iron-limited media respectively. The Blank strain was set as controls.

Usage and Applications

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 985
    Illegal BsaI.rc site found at 1907
    Illegal BsaI.rc site found at 2634