Difference between revisions of "Part:BBa K1401008"

Latest revision as of 00:35, 14 October 2014

Tandem Promoter pBad-pTet

This tandem promoter is a combination of pBAD-pTetR and was created using the MoClo assembly method. This is a Level 0 MoClo part with flanking sites A on the 5' side and site B on the 3' side of the part. The fusion site letters refer to 4bp fusion sites: A = GGAG; B = TACT; C = AATG; D = AGGT; E = GCTT; F = CGCT; G = TGCC; H = ACTA. This tandem promoter is in pSB1C3.

The promoter is supposed to be induced by anhydrotetracycline (aTc) and arabinose.

In order to test this tandem promoter, we built a basic transcriptional unit with red fluorescent protein reporter. We used the E. coli DH5-alpha Pro strain for this study, which has tetR, lacI, and araC all constitutively expressed in its genome. We induced the E. coli with aTc alone (red circles), arabinose alone (blue circles), and then both aTc and arabinose together (purple circles).

As shown below, the aTc induction worked while the arabinose failed to induce the expression of RFP. This tandem promoter only functions halfway as expected and is induced only with aTc alone. We are planning another set of experiments to explore a higher range of arabinose to see if we can induce pBAD. Fluorescence drops at 5000 and 10000 ng/mL aTc due to a toxicity effect where the E. coli cells died.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 243
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 78
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI site found at 60

@@ Line 6: / Line 6: @@
 The promoter is supposed to be induced by anhydrotetracycline (aTc) and arabinose.
-In order to test this tandem promoter, we built a basic transcriptional unit with red fluorescent protein reporter. We used the ''E. coli'' DH5-alpha Pro strain for this study, which has ''tetR'', ''lacI'', and ''araC'' all constitutively expressed in its genome. We induced the ''E. coli'' with aTc alone (red circles), arabinose alone (blue circles), and then both aTc and arabinose together (purple circles) to see if we could determine if one promoter was dominant over the other. As shown below, the aTc induction worked while the arabinose failed to induce the expression of RFP.
+In order to test this tandem promoter, we built a basic transcriptional unit with red fluorescent protein reporter. We used the ''E. coli'' DH5-alpha Pro strain for this study, which has ''tetR'', ''lacI'', and ''araC'' all constitutively expressed in its genome. We induced the ''E. coli'' with aTc alone (red circles), arabinose alone (blue circles), and then both aTc and arabinose together (purple circles).
-As shown below, this tandem promoter only functions halfway as expected and is induced only with aTc alone. We are planning another set of experiments to explore a higher range of arabinose to see if we can induce pBAD. Fluorescence drops at 5000 and 10000 ng/mL aTc due to a toxicity effect where the ''E. coli'' cells died.
+As shown below, the aTc induction worked while the arabinose failed to induce the expression of RFP. This tandem promoter only functions halfway as expected and is induced only with aTc alone. We are planning another set of experiments to explore a higher range of arabinose to see if we can induce pBAD. Fluorescence drops at 5000 and 10000 ng/mL aTc due to a toxicity effect where the ''E. coli'' cells died.
 [[File:K1401008_flow.png|center|450px]]