Difference between revisions of "Part:BBa K337008"
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<partinfo>BBa_K337008 parameters</partinfo>
 
<partinfo>BBa_K337008 parameters</partinfo>
 
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=== Characterization of synthetic microRNA binding site patterns against endogenous miR122 ===
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We used the miMeasure plasmid with the BB-2 standard to characterise binding sites for the endogenously expressed miR122. We cloned two different synthetic microRNA binding site patterns into our miMeasure construct plasmid:3.1 [https://parts.igem.org/Part:BBa_K337008 BBa_K337008] and 1.3 [https://parts.igem.org/Part:BBa_K337000 BBa_K337000]. As the binding site is inserted downstream of EGFP, a regulation of EGFP expression is to be expected. miMeasure normalizes knockdown of the green fluorescent protein (EGFP) to the unregulated blue fluorescent protein (EBFP2). By calculating the ratio of EGFP to EBFP2 we analyzed the knockdown percentage of the characterized binding site patterns.
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We tested these constructs in four different set-ups with three different cell lines:
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*HeLa cells, which does not express miR122 endogenously
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*HeLa cells cotransfected with miR122
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*HuH7 cells, liver cells known to express miR122, and
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*HepG2 cells, liver cells known to express low amounts of miR122 {{HDref|Douglas, 2010}}
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EGFP to EBFP2 ratios were meassured with flow cytometry and microscopy. Measurement results for the four cell lines and the binding sites 3.1 [https://parts.igem.org/Part:BBa_K337008 BBa_K337008] and 1.3 [https://parts.igem.org/Part:BBa_K337000 BBa_K337000] are shown in '''Fig. 4''' for all 4 cell line setups. As expected, no downregulation of EGFP expression was meassured in HeLa cells. Similarly, no downregulation was observed in HepG2 cells. Both the construct 1.3 (containing 2 perfect binding sites with the extra 10 bp spacer in between) and 3.1 (containing 3 perfect binding sites) managed to increase the downregulation effect of EGFP in Huh7 cells and in HeLa cells cotransfected with miR122 in comparison to the single perfect binding site. 
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[[Image:Download-1.jpg|thumb|center|800px|'''Figure 4: miMeasure in four different set-ups with three different cell lines''' The construct containing synthetic microRNA binding site patterns against endogenous miR122 transfected into different cell lines. The EGFP/EBFP2 ratio for the construct containing no binding site was set to one.]]

Revision as of 01:04, 28 October 2010

hsa-miR-122 Binding site pattern (2BS) with randomised nt9-12

hsa-miR-122 binding site pattern, consisting of 2 binding sites (BS). The BS are seperated by sequences of 30 base pairs, which are inert in terms of miRNA binding. Additionally, half of the separation sequence is either in front or the end of the whole pattern, to be unconnected to standard restriction sites (BB-2, RFC12).

The binding sites were created by using oligos where the reverse complemented nucleotides 9-12 of the corresponding miRNA were randomised:

CAAACACCATNNNNACACTCCA


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]




Characterization of synthetic microRNA binding site patterns against endogenous miR122

We used the miMeasure plasmid with the BB-2 standard to characterise binding sites for the endogenously expressed miR122. We cloned two different synthetic microRNA binding site patterns into our miMeasure construct plasmid:3.1 BBa_K337008 and 1.3 BBa_K337000. As the binding site is inserted downstream of EGFP, a regulation of EGFP expression is to be expected. miMeasure normalizes knockdown of the green fluorescent protein (EGFP) to the unregulated blue fluorescent protein (EBFP2). By calculating the ratio of EGFP to EBFP2 we analyzed the knockdown percentage of the characterized binding site patterns.

We tested these constructs in four different set-ups with three different cell lines:

  • HeLa cells, which does not express miR122 endogenously
  • HeLa cells cotransfected with miR122
  • HuH7 cells, liver cells known to express miR122, and
  • HepG2 cells, liver cells known to express low amounts of miR122 Template:HDref

EGFP to EBFP2 ratios were meassured with flow cytometry and microscopy. Measurement results for the four cell lines and the binding sites 3.1 BBa_K337008 and 1.3 BBa_K337000 are shown in Fig. 4 for all 4 cell line setups. As expected, no downregulation of EGFP expression was meassured in HeLa cells. Similarly, no downregulation was observed in HepG2 cells. Both the construct 1.3 (containing 2 perfect binding sites with the extra 10 bp spacer in between) and 3.1 (containing 3 perfect binding sites) managed to increase the downregulation effect of EGFP in Huh7 cells and in HeLa cells cotransfected with miR122 in comparison to the single perfect binding site.


Figure 4: miMeasure in four different set-ups with three different cell lines The construct containing synthetic microRNA binding site patterns against endogenous miR122 transfected into different cell lines. The EGFP/EBFP2 ratio for the construct containing no binding site was set to one.