Part:BBa_K1889001
Constitutive Promoter Expressing 4CL gene
Background
K1889001 encloses promoter J23100, RBS B0034, gene K1129042 and a double terminator B0015. J23100 is a from the constitutive promoter family. K1129042 is designed by iGEM13_British_Columbia. K1129042 is a coding region encoded 4-coumarate-CoA ligase (4CL). 4CL is an enzyme catalyzes the ligation of a CoA group to the end of the molecule. The addition of this CoA provides better kinetics for a variety of downstream reactions. 4CL is also a member of the phenylpropanoid pathway and is found in a wide variety of organisms that partake in the degradation of lignin. Here we harness the 4CL to provide better kinetics for reduction of cinnamic acid to cinnamaldehyde. In the figure below figure we coupled the activity of PAL with a cinnamic acid ligase and a Cinnamoyl Co A reductase to yield cinnamaldehyde from the aromatic amino acid phenylalanine.
Assay of pure chemical of cinnamaldehyde
The conducted assays were to test whether the chemicals are attractant or repellent to C. elegans. And we use Response index to describe whether the chemical is an attractant or repellent to C. elegans. First, we mark the substance loading spot on the lid where we put volatile substances. As cinnamaldehyde is a volatile substance, we conducted the volatile test. For volatile test, we pipetted 2 ul of tested volatile substance on the loading spot in the two ‘T’ areas on the lid. And load the same volume of water on the ‘C’ spots on the lid. Then, we put the assay plate aside and leave it undisturbed for 30 minutes. After 30 minutes, we obtained the total number of worms in the ‘T’ areas and ‘C’ areas. The response index was calculated by formula “ T minus C over T plus C“ . T is the number of worms in the ‘T’ areas. And C is the number of worms in the ‘C’ areas, which means the number of worms that entered the areas by chance . The denominator ‘T plus C’ represents the total number of worms in the counted areas, that is inside the 4 circles. The numerator T minus C is the number of worms which made response due to the effect of the tested substances.
The index value will range between -1 to 1. If the index is negative, it means the tested chemical is repellent; in contrast, positive index means the tested chemical is attractant. We tested our assay and response index by chemical diacetyl, a known strong attractant to C. elegans. And the result is promising that the Response index of volatile is close to 1.0, which means that assay and response index can show diacetyl is a strong attractant. We also tried using water as negative control for the assay. The Response index is 0.0x. Which means the assay can show that the worms has no significant response, neither attraction or repulsion, to water. The result is that cinnamaldehyde’s index is -0.81 when concentration is 1, -0.8 at 1/10, -0.82 at 1/100, -0.5 at 1/1000, -0.13 at 1/10000 and -0.1 at 1/100000. With the evidence provided by the tests, the results are clearly shown. Cinnamaldehyde is a strong repellent.
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Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NheI site found at 1147 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1714
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1346
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