Difference between revisions of "Part:BBa K1084104"
 
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Synthetic RBS with Enhancer sequence and 8 bp SD sequence
 
Synthetic RBS with Enhancer sequence and 8 bp SD sequence
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We made these constructs to measure translation efficiency of our RBSs (SD2: BBa_K1084101, SD4: BBa_K1084102, SD6: BBa_K1084103, SD8: this part). We used TetR repressible promoter (pTet: BBa_R0040), LacZ&alpha; (BBa_I732006) and double terminator (dT: BBa_B0015).
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  positive control<dd>pTet-B0034-LacZ&alpha;-dT</dd>
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  negative control<dd>No insert</dd>
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  SD2<dd>pTet-SD2-LacZ&alpha;-dT (BBa_K1084121)</dd>
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  SD4<dd>pTet-SD4-LacZ&alpha;-dT (BBa_K1084122)</dd>
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  SD6<dd>pTet-SD6-LacZ&alpha;-dT (BBa_K1084123)</dd>
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  <dt>SD8</dt><dd>pTet-SD8-LacZ&alpha;-dT (BBa_K1084124)</dd>
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We moved our constructs to pSB3K3 plasmid, cultured for 9 hrs in 5 mL LB media round tubes, and performed &beta;-Galactisidase assay. Reaction time was 2 hrs. We did this assay for 24 times.
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<div class="fig fig800">
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  <img src="https://static.igem.org/mediawiki/2013/f/f3/HokkaidoU_RBS_results1_800.png">
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  <div>fig.1: Picture of &beta;-Galactosidase assay.  orange: strong LacZ&alpha; expression, yellow: weak expression.</div>
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<div class="fig fig800">
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  <img src="https://static.igem.org/mediawiki/2013/3/3d/HokkaidoU_RBS_results2_800.png">
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  <div>fig.2: LacZ&alpha; expression. x axis: sample name, y axis: standardized expression (positive control = 1.0), bar: standard error.</div>
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LacZ&alpha; protein hydrolyses substrate (Chlorophenol red-&beta;-D-galactopyranoside), which is yellow. Product of this reaction has red color. Therefore, solution will turn deep orange if LacZ&alpha; expression is strong (fig.1).
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We measured absorbance of catalytic reaction solution at 595 nm, standardized using positive control and made into graph (fig.2).
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Construct with SD4 showed the strongest LacZ&alpha; activity. Second strongest was the SD8, followed by SD6. SD2 had the weakest activity. There is a significant difference in translation efficiency of our RBSs. Translation efficiency of SD4 and SD8 is the same as BBa_B0034, which is most used RBS.
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 13:13, 2 October 2013

SD8

Synthetic RBS with Enhancer sequence and 8 bp SD sequence


We made these constructs to measure translation efficiency of our RBSs (SD2: BBa_K1084101, SD4: BBa_K1084102, SD6: BBa_K1084103, SD8: this part). We used TetR repressible promoter (pTet: BBa_R0040), LacZα (BBa_I732006) and double terminator (dT: BBa_B0015).

positive control
pTet-B0034-LacZα-dT
negative control
No insert
SD2
pTet-SD2-LacZα-dT (BBa_K1084121)
SD4
pTet-SD4-LacZα-dT (BBa_K1084122)
SD6
pTet-SD6-LacZα-dT (BBa_K1084123)
SD8
pTet-SD8-LacZα-dT (BBa_K1084124)

We moved our constructs to pSB3K3 plasmid, cultured for 9 hrs in 5 mL LB media round tubes, and performed β-Galactisidase assay. Reaction time was 2 hrs. We did this assay for 24 times.

fig.1: Picture of β-Galactosidase assay. orange: strong LacZα expression, yellow: weak expression.
fig.2: LacZα expression. x axis: sample name, y axis: standardized expression (positive control = 1.0), bar: standard error.

LacZα protein hydrolyses substrate (Chlorophenol red-β-D-galactopyranoside), which is yellow. Product of this reaction has red color. Therefore, solution will turn deep orange if LacZα expression is strong (fig.1).

We measured absorbance of catalytic reaction solution at 595 nm, standardized using positive control and made into graph (fig.2). Construct with SD4 showed the strongest LacZα activity. Second strongest was the SD8, followed by SD6. SD2 had the weakest activity. There is a significant difference in translation efficiency of our RBSs. Translation efficiency of SD4 and SD8 is the same as BBa_B0034, which is most used RBS.


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]