Difference between revisions of "Part:BBa I0500:Experience"
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[[Image:Pbadbgal.jpg|left|thumb|400px|'''Beta-galactosidase activity of cultures with different concentrations of arabinose increases with raising the arabinose concentration.''']]
 
[[Image:Pbadbgal.jpg|left|thumb|400px|'''Beta-galactosidase activity of cultures with different concentrations of arabinose increases with raising the arabinose concentration.''']]
  
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The tested cultures incubated at different concentrations of arabinose (i.e. 0%, 0.001%, 0.0025%, 0.005%, 0.01%, 0.025%, 0.05%, 0.1%, 0.25%, 0.5%, 0.75% and 1%) have shown a trend of increasing beta-galactosidase activity with raising arabinose concentration. The activity was highest at 1% added arabinose, which implies the promotor should be induced by this arabinose concentration for reaching maximum expression of the regulated protein. However, some beta-galactosidase activity was observed even at 0% added arabinose, which indicates slight leakage of the pBAD promoter.
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== pBAD/araC_lacZ_DTER construct ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K323133 Part:Bba_K323133]).  ==
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This part includes a beta-galactosidase (lacZ) gene, the expression of which is regulated by the pBAD promoter ([https://parts.igem.org/wiki/index.php/Part:BBa_I0500 Part:Bba_I0500]). This construct was designed for the sole purpose of characterising the pBAD promoter with the beta-galactosidase assay (for a detailed description of the method see the 2010 iGEM team Slovenia wiki).
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The pBAD promoter is an arabinose inducible promoter. Different concentrations of arabinose were added to cultures of bacteria containing this BioBrick part, and beta-galactosidase activity was measured to determine the optimal concentration of arabinose for maximum activity of the promoter.
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For results see ([https://parts.igem.org/wiki/index.php/Part:BBa_I0500 Part:Bba_I0500]).
 
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Revision as of 17:11, 26 October 2010

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_I0500

  • When grown with 0.2% arabinose, promoter is weak-medium. [jb, 5/24/04] Part may not be compatible with MC4100 as cell line is araD 139.
  • MC4100 is not a good chassis for operating BBa_I0500 (pBad promoter). The feed-forward regulation of the endogenous promoter controlling expression of the arabinose transporter prevents linear induction with increasing arabinose concentration. (Engineered strain from Keasling's lab, used by jrk for operation of the screening plasmid.) [cconboy 04]
  • Observations of induced expression of GFP (BBa_E0840) are consistent with previous comments about weak promoter signal induction by jb 5/24/04. [melissali, Berkeley iGEM 2005]

PC and AraC are located on the complementary strand, reading right to left as written.

  • At least one registry stock contains a deletion of the C at base 1194. This is after the transcriptional start but before the translation start, so it may not be significant. Parts with this mutation have been qualitatively observed to function normally.

User Reviews

2010 iGEM team Slovenia further characterized the pBAD/araC promoter (Part:Bba_I0500) using lacZ gene that encodes for beta-galactosidaze enzyme (Part:Bba_K323133). When incubating E.coli cultures containing the lacZ under pBAD/araC promoter, increasing concentrations of arabinose leads to higer promoter activity that results in higher amounts and subsequently higer activity of beta-galactosidase enzyme as seen from figure below. The resulting graph shows a trend of increasing beta-galactosidase activity over increasing concentrations of arabinose.


Beta-galactosidase activity of cultures with different concentrations of arabinose increases with raising the arabinose concentration.


The tested cultures incubated at different concentrations of arabinose (i.e. 0%, 0.001%, 0.0025%, 0.005%, 0.01%, 0.025%, 0.05%, 0.1%, 0.25%, 0.5%, 0.75% and 1%) have shown a trend of increasing beta-galactosidase activity with raising arabinose concentration. The activity was highest at 1% added arabinose, which implies the promotor should be induced by this arabinose concentration for reaching maximum expression of the regulated protein. However, some beta-galactosidase activity was observed even at 0% added arabinose, which indicates slight leakage of the pBAD promoter.


pBAD/araC_lacZ_DTER construct (Part:Bba_K323133).

This part includes a beta-galactosidase (lacZ) gene, the expression of which is regulated by the pBAD promoter (Part:Bba_I0500). This construct was designed for the sole purpose of characterising the pBAD promoter with the beta-galactosidase assay (for a detailed description of the method see the 2010 iGEM team Slovenia wiki).


The pBAD promoter is an arabinose inducible promoter. Different concentrations of arabinose were added to cultures of bacteria containing this BioBrick part, and beta-galactosidase activity was measured to determine the optimal concentration of arabinose for maximum activity of the promoter.


For results see (Part:Bba_I0500). UNIQ82098676fc9d249e-partinfo-00000000-QINU


•••••

Antiquity

This review comes from the old result system and indicates that this part worked in some test.

iGEM Groningen 2009

The sequence was listed as inconsistent, and the ligations of parts behind the promoter failed. Restriction of isolated plasmids showed fragments of unexpected sizes.

UNIQ82098676fc9d249e-partinfo-00000003-QINU