Difference between revisions of "Part:BBa K3866006"

Revision as of 14:56, 25 September 2021

ParaBAD-ackA-Terminator

Usage and Biology

This TU includes the AckA gene placed under the control of the arabinosed-induced promoter. The inducible promoter is used because cloning wasn't done. After the constitutive expression, the metabolism went out of balance so no colonies survied to be chosen for screening.

Design Notes

The coding sequence was domesticated . We removed BsmBI and BsaI sites in order to be compatible with GoldenBraid and MoClo. The sequence is cloned in seva a2 vector and has overhangs compatible for GoldenBraid cloning.

Figure 1. The level a module of the Actetate Production : a2:ParaBAD:RBS-AckA-Double terminator

Verification of Cloning

Fig.2:: (U=Uncut , C= Cut)Positive clone: 6,Seva a2 arac ACK. Expected bands 4601, 853

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 1148
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 983
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI site found at 965

References

Dittrich, C. R., Bennett, G. N., & San, K. Y. (2005). Characterization of the acetate-producing pathways in Escherichia coli. Biotechnology progress, 21(4), 1062–1067. https://doi.org/10.1021/bp050073s

@@ Line 10: / Line 10: @@
 The coding sequence was domesticated . We removed BsmBI and BsaI sites in order to be compatible with GoldenBraid and MoClo. The sequence is cloned in seva a2 vector and has overhangs compatible for GoldenBraid cloning.
-[[Image:T--Thessaly--FFAR2-VTAIL-PHOT.png|900px|thumb|none|<I><b>Figure 1.</b> The level B module of GPCR-Tango Module : a1R:ParaBAD:RBS-FFAR2:V2tail:TCS-Lac-Double terminator </i>]]
+[[Image:T--Thessaly--arac-ack-term.png|900px|thumb|none|<I><b>Figure 1.</b> The level a module of the Actetate Production : a2:ParaBAD:RBS-AckA-Double terminator </i>]]
 ===Verification of Cloning===
-[[File:T--Thessaly--FFAR2-LACI-digestion.png|700px|thumb|none|<i><b>Fig.2:</b>: (U=Uncut , C= Cut) Restriction digestion a1R:ParaBAD:RBS-FFAR2:V2tail:TCS-Lac-Double terminato (C1a-C4b) with : BamHI(C1a-C4a) , Expected bands : 2847+2225 bp , EcoRV (C2a-C2b) ,Expected bands : 3587 bp + 2845 bp, Positive result: C1,C2,C3,C3 (C1a and C1b is the same sample etc)</i>]]
+[[File:T--Thessaly--arac-ackgel.png|700px|thumb|none|<i><b>Fig.2:</b>: (U=Uncut , C= Cut)Positive clone: 6,Seva a2 arac ACK. Expected bands 4601, 853</i>]]