Difference between revisions of "Part:BBa K574004"

Latest revision as of 13:33, 21 October 2019

3OC12HSL regulated by TetR

TetR represses the processing of sending 3OC12HSL, and aTc, which can bind TetR, can act as an activator in this condition.

Measuring Expression: Alma 2019

To test the expression and activity of BBa_K204047, K574004, and K1692023, we (the Alma College iGEM team) took the following steps. Equal volumes of mid-log cells were resuspended in fresh LB + Chloramphenicol. These cultures were incubated for 6 hours at 37C in a tube rotator. Cells were pelleted by centrifugation - the supernatant was saved for detection of AHL molecules (all those synthesized during the incubation).

From the remaining cell pellet, proteins were isolated using the B-PER reagent (Thermo-Scientific). Soluble and Insoluble proteins were then run on an SDS PAGE gel. The gel was stained with coomaise, and density analsyis was performed with ImageJ.

This is a representative image from several independent replicates. S = Soluble protein fraction, I = Insoluble protein fraction. The negative control used in this particular replicate was an uninduced culture of strain BBa_T9002. Density analysis of this image (found on our Wiki) confirm that there are no significant bands or signal for the AHL synthase BioBrick cultures above background in the 20-25 kDa range, which is where the AHL synthases would be expected to be found.

From these results we were barely able to detect any expression of the AHL synthase genes - proteins such as LuxI and LasI should be present as bands slightly below the 25 kDa marker. However the supernatants from similar cultures did display AHL when tested with the biosensor strain BBa_T9002. For an exmaple of this data, please see BBa_K1692023. Thus, we conclude that the proteins are present but at very low quantities - likely a result of the degrons present, leading to a very short half-life.

At the same time as performing these experiments, we were able to detect protein expression for reporters such as RFP (BBa_J04450) and GFP (BBa_K515005), suggesting that our technique and experimental setup was not responsible for our inability to see expression of the AHL synthease genes.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 744
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 306
1000
COMPATIBLE WITH RFC[1000]

@@ Line 4: / Line 4: @@
 TetR represses the processing of sending 3OC12HSL, and aTc, which can bind TetR, can act as an activator in this condition.
-<!-- Add more about the biology of this part here
+===Measuring Expression: Alma 2019===
-===Usage and Biology===
+To test the expression and activity of [https://parts.igem.org/Part:BBa_K204047 BBa_K204047], K574004, and [https://parts.igem.org/Part:BBa_K1692023 K1692023], we (the Alma College iGEM team) took the following steps. Equal volumes of mid-log cells were resuspended in fresh LB + Chloramphenicol. These cultures were incubated for 6 hours at 37C in a tube rotator. Cells were pelleted by centrifugation - the supernatant was saved for detection of AHL molecules (all those synthesized during the incubation).
+From the remaining cell pellet, proteins were isolated using the B-PER reagent (Thermo-Scientific). Soluble and Insoluble proteins were then run on an SDS PAGE gel. The gel was stained with coomaise, and density analsyis was performed with ImageJ.
+[[File:T--Alma--AHLsynthaseExpression1.png]]
+This is a representative image from several independent replicates. S = Soluble protein fraction, I = Insoluble protein fraction. The negative control used in this particular replicate was an uninduced culture of strain BBa_T9002. Density analysis of this image (found on our Wiki) confirm that there are no significant bands or signal for the AHL synthase BioBrick cultures above background in the 20-25 kDa range, which is where the AHL synthases would be expected to be found.
+From these results we were barely able to detect any expression of the AHL synthase genes - proteins such as LuxI and LasI should be present as bands slightly below the 25 kDa marker. However the supernatants from similar cultures did display AHL when tested with the biosensor strain [https://parts.igem.org/Part:BBa_T9002 BBa_T9002]. For an exmaple of this data, please see [https://parts.igem.org/Part:BBa_K1692023 BBa_K1692023]. Thus, we conclude that the proteins are present but at very low quantities - likely a result of the degrons present, leading to a very short half-life.
+At the same time as performing these experiments, we were able to detect protein expression for reporters such as RFP (BBa_J04450) and GFP (BBa_K515005), suggesting that our technique and experimental setup was not responsible for our inability to see expression of the AHL synthease genes.
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