Measurement

Part:BBa_K654052

Designed by: Sarah Allred   Group: iGEM11_Utah_State   (2011-09-26)
Revision as of 19:12, 28 September 2011 by Catramp (Talk | contribs)

Promoter/RBS Tester sigA Reference Dual Luciferase Assay

This BioBrick is part of the Dual Luciferase Testing system. It contains the Firefly Luciferase coding region, BioBrick Standard Terminator, sigA Synechocystis Reference Promoter and RBS, Renilla Luciferase coding region, and the standard BioBrick terminator.

This part used to determine the strength of the promoter of interest relative to the sigA Synechocystis reference promoter (for full details on use and more about the system in general, see below)

Dual Luciferase Testing System

The Dual Luciferase Testing system is a promoter and ribosome binding site (RBS) characterization and measurement system that controls for several testing variables that older systems do not. The system consists of two luciferase genes, Firefly Luciferase (BBa_I712019) and Renilla Luciferase (BBa_J52008) that bioluminesce at two distinct wavelengths (560 nm and 475 nm, respectively). The parts in this system are designed to allow quick and easy assembly of measurement devices and to provide the ability to customize the system to your organism or promoter reference of choice (full details are at the bottom of this description).

The expression level of your promoter of interest (cloned in front of Firefly Luciferase) is then compared to the expression level of the reference promoter in front of Renilla Luciferase (this promoter is generally the Anderson 1.0 reference promoter BBa_J23100, but can be changed to other promoters for use in other species). The ratio of Firefly to Renilla expression indicates the relative strength of the promoter of interest. To control for differences in the expression of the two genes due to codon bias and other differences, the Firefly:Renilla ratio of the promoter of interest to the reference promoter is compared to a Dual Luciferase construct with the reference promoter controlling expression of both luciferase genes. This adjusted ratio gives the true expression level of the promoter.


This system has the following advantages over the commonly used RFP expression measurement system:

  1. Copy number control – Plasmids, and particularly high copy number plasmids, generally do not have a single set number of copies in a cell, but can vary in a wide range. The iGEM standard plasmids are high copy number plasmids that can have between 100-300 copies in a cell (Example: pSB1C3). By having both the promoter of interest and the reference promoter controlled genes on the same plasmid (rather than in two different cell cultures like the RFP system) the genes are present in equal numbers, controlling this variable factor.

  2. Cell cycle phase control – Since both genes are present in each cell, the reference will still be a viable control regardless of cell cycle phase. In the RFP system, if the two cultures containing the testing promoter and the reference promoter were in different phases, the levels of expression would not be comparable and the promoter strengths would be incorrectly calculated.

  3. Cell density control – Since both genes are present in each cell in the culture, the density of the culture does not affect the ratio of the promoter’s expression. In the RFP system, if cell densities were different between the control and reference cultures, the expression levels of the promoters would be incorrectly measured (the more dense culture having a higher apparent expression level), and the promoter strength calculation would be inaccurate.

This system does require some additional supplies that the RFP system does not, but these additional supplies are not prohibitively difficult to obtain:

  1. Luminometer – Since luciferase proteins convert chemical energy to light, standard spectrophotometers, which rely on the amount of light that a sample absorbs, will not correctly measure the light output. Luminometers are designed to measure emitted light levels at specific wavelengths, and are likely to be found on most college campuses.

  2. Chemical Energy (Luciferin) – Luciferase proteins produce light by causing chemical conversions of chemicals called luciferins. These luciferins need to be added to lysed cells solutions in order to measure the expression of the proteins, unlike RFP which can be measured within living cells and with only the addition of light of the proper wavelength. There are several commercial kits available specifically for dual luciferase assays including the [http://www.promega.com/products/reporter-assays-and-transfection/reporter-assays/dual_luciferase-reporter-assay-system/ Promega Dual Luciferase Reporter Assay System].

BioBrick Parts in this Set

The set of BioBrick parts in this system allows for quick and easy assembly of promoter and RBS strengths, and makes it easy for custom promoters and RBSs to be used as reference standards, allowing this system to be used in a variety of species. The following BioBricks are part of this set, and utilize a group of E. coli and Synechocystis sp. PCC 6803 reference promoters (see the assembly section for how to assemble your measurement construct):

BBa_K654033 - BBa_K654055, BBa_K654092 - BBa_K654094


Assembling Your Measurement Device (using RF 10 or RF 23):

  1. Testing a promoter strength against an existing reference standard in the set:

    1. Digest BioBrick vector containing your promoter of interest with SpeI and PstI
    2. Digest Promoter Tester with the reference standard you want to use with XbaI and PstI (Anderson 1.0 E. coli reference: BBa_K654045, psbA2 Synechocystis reference with Native RBS: BBa_K654048, psbA2 Synechocystis reference with Consensus RBS: BBa_K654054, sigA Synechocystis reference with Native RBS: BBa_K654051, sigA Synechocystis reference with Consensus RBS: BBa_K654055)
    3. Ligate the Promoter Testing part into the promoter’s vector.
    4. Transform into E. coli (or another desired species)
    5. Transform the Promoter Control into E. coli. This promoter control has the reference promoter in front of both luciferase genes, to control for differences in expression due to the codon usage of the two luciferase genes (Anderson 1.0 E. coli Control: BBa_K654047, psbA2 Synechocystis control: BBa_K654050, sigA Synechocystis control: BBa_K654053)
    6. Obtain the Firefly/Renilla Ratio of the promoter of interest to the reference standard using the protocol of the assay kit ([http://www.promega.com/products/reporter-assays-and-transfection/reporter-assays/dual_luciferase-reporter-assay-system Promega Kit Link]), and divide this ratio by the Promoter Control Firefly/Renilla ratio to adjust for codon usage differences.

  2. Testing a promoter and ribosome binding site (or just a ribosome binding site) strength against an existing reference standard in the set:

    1. If testing a ribosome binding site of interest only, for the following steps, simply substitute the reference standard promoter you are going to be comparing against in for the promoter of interest (this will make the RBS the only difference between the two luciferase expression rates)
    2. Clone the RBS to test downstream of the promoter of interest
    3. Digest BioBrick vector containing your promoter and RBS of interest with SpeI and PstI
    4. Digest Promoter/RBS Tester with the reference standard you want to use with XbaI and PstI (Anderson 1.0 E. coli reference: BBa_K654046, psbA2 Synechocystis reference: BBa_K654049, sigA Synechocystis reference: BBa_K654052)
    5. Ligate the Promoter Testing part into the promoter’s vector.
    6. Transform into E. coli (or another desired species)
    7. Transform the Promoter Control into E. coli. This promoter control has the reference promoter in front of both luciferase genes, to control for differences in expression due to the codon usage of the two luciferase genes (Anderson 1.0 E. coli Control: BBa_K654047, psbA2 Synechocystis control: BBa_K654050, sigA Synechocystis control: BBa_K654053).
    8. Obtain the Firefly/Renilla Ratio of the promoter of interest to the reference standard using the protocol of the assay kit, and divide this ratio by the Promoter Control Firefly/Renilla ratio to adjust for codon usage differences. ([http://www.promega.com/products/reporter-assays-and-transfection/reporter-assays/dual_luciferase-reporter-assay-system Promega Kit Link])

Making a custom reference:

  1. If you want to just add a different reference promoter and keep the Reference RBS:

    1. Clone the RBS + Renilla Luciferase + Terminator part (E. coli 1.0 RBS: BBa_K654092, Synechocystis Native RBS: BBa_K654093, Synechocystis Consensus RBS: BBa_K654094) downstream of your desired reference promoter.
    2. Clone the desired Firefly luciferase construct upstream of the construct from Step 1. If you are testing only promoters you will want a RBS + Firefly Luciferase + Terminator part (E. coli 1.0 RBS: BBa_K654042, Synechocystis Native RBS: BBa_K654043, Synechocystis Consensus RBS: BBa_K654044) and if you are going to test your own promoter and RBS, you want the Firefly + Terminator only part (BBa_K654033).
    3. You will also need to prepare a Promoter Control construct (with the same reference promoter in front of the Firefly and Renilla luciferases) to control for codon usage differences between the genes. This is done by cloning the reference promoter you want upstream of a RBS + Firefly Luciferase + Terminator part (E. coli 1.0 RBS: BBa_K654042, Synechocystis Native RBS: BBa_K654043, Synechocystis Consensus RBS: BBa_K654044). Then you clone this Reference Firefly construct you just made upstream of the reference Renilla construct you made in Step 1.

  2. If you want to just add a different reference promoter AND reference RBS:

    1. Clone the desired reference RBS downstream of the desired reference promoter. (If the promoter and RBS are too small to easily clone, you can attach the luciferase + terminator parts BBa_K654033 and BBa_K654034 downstream of the RBS, and then clone these RBS + Luciferase + Terminator constructs downstream of the promoter, the following steps may change a bit if you do it this way)
    2. Clone the Renilla Luciferase + Terminator (BBa_K654034) part downstream of your desired reference promoter and RBS construct.
    3. Clone the desired Firefly luciferase construct upstream of the construct from Step 2. If you are testing only promoters you will want a RBS + Firefly Luciferase + Terminator part (E. coli 1.0 RBS: BBa_K654042, Synechocystis Native RBS: BBa_K654043, Synechocystis Consensus RBS: BBa_K654044) and if you are going to test your own promoter and RBS, you want the Firefly + Terminator only part (BBa_K654033).
    4. You will also need to prepare a Promoter Control construct (with the same reference promoter in front of the Firefly and Renilla luciferases) to control for codon usage differences between the genes. This is done by cloning the reference promoter and RBS you want upstream of the Firefly Luciferase + Terminator part (BBa_K654033). Then you clone this Reference Firefly construct you just made upstream of the reference Renilla construct you made in Step 2.

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
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 2412
    Illegal SapI.rc site found at 808


[edit]
Categories
Parameters
None