Difference between revisions of "Part:BBa K4129103"

Revision as of 21:05, 10 October 2022

The synthetic transcription factor, FunsTF05 (LexA-LL-Hmox1-VP16-SV40)

FunsTF05 is a synthetic transcription factor (sTF). FunsTF05 should function as a transcription factor that can initiate transcription of the 6xLexO minimal promoter (BBa_K4129115). This sTF will be the sensing part of the biosensor.

FunsTF05 is a fusion protein consisting of the DNA-binding domain: LexA, ligand sensing domain: Hmox1, transactivation domain; VP16 and the nuclear localization signal (NLS) SV40. The linker between LexA and Hmox1 was a longer version (Ottoz et. al (2014) compared to sBAD, which was the reference sTF (Castaño-Cerezo et. al (2020)).

LexA is a repressor that regulates the SOS response in E. coli (Radman. 1975). LexA binds to a specific DNA motif, LexO (Erill. et al (2003)), and it is the DNA binding domain that interacts with LexO that is used in FunsTF05. Hmox1 is the human heme oxygenase 1, which is the enzyme that initiates cleavage of heme (Tenhunen et al. (1969)). This function is not of our interest, but it was computationally shown that furfural binds to hmox1 (Santhakumar et al (2021)).

Viral Protein 16 (VP16) from herpes simplex virus type 1 is a transcription factor that uses a transactivation domain to recruit the RNA polymerase II (Hirai et al. (2010)).The NLS SV40 is a small peptide sequence of PKKKRKV that enables transport of the protein to the nucleus (Garcia-Bustos et. al (1991)).

Characterization

The functionally of sTF05 were tested by measuring the fluorescence of an A. niger carring sTF05 and the mCherry reporter (BBa_K4129123). The A. niger is grown on solid media plates. The plates contained either minimal media, minimal media with mM benzoic acid or MM with 0.6 g/L furfural.

The fluorescence was assessed, after four days of incubation at 30℃, using the Vilber Fusion FX imager system. A grey-white intensity scale was used to present the measured fluorescence. The exposure time was normalised to the fluorescences from genomic integrated BBa_K3046004, and this enabled comparison between plates. The exposure time used was 1.04 seconds. It is observed that genomic integrated BBa_K3046004 displays fluorescence and the negative control of BBa_K4129025 did not. It should be noted that no growth was observed on the plates containing 0.6 g/L furfural (figure 1).

Figure 1: Pictures of fluorescent A. *niger*, which carries either BBa_K4129025 or genome integrated BBa_K3046004. The picture are taken with 1.04 seconds exposure time. The A. *niger* is grown on plates containing minimal media (MM), MM with 2 mM benzoic acid or MM with 0.6 g/L furfural. The fluorescent is depicted as grey-white intensity..

The observed fluorescence of FunsTF05 was greater than that of genomic integrated BBa_K3046004 on the different plates (figure 2). BBa_K3046004 is a strong constitutive promoter in A. niger, and this emphasise the stregth of FunsTF05.

The observed fluorescence of FunsTF05 did not differ too much between plates, which indicate FunsTF05 constitutively expressed mCherry (BBa_K4129123) (figure 2).

Figure 2: Pictures of fluorescent A. *niger*, which carries FunsTF05. The pictures are taken with 1.04 seconds exposure time. The A. *niger* is grown on plates containing minimal media (MM), MM with 2 mM benzoic acid or MM with 0.6 g/L furfural. The fluorescent is depicted as grey-white intensity.

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 945
Illegal BamHI site found at 607
Illegal XhoI site found at 800
Illegal XhoI site found at 1237
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 15: / Line 15: @@
 The functionally of sTF05 were tested by measuring the fluorescence of an <i>A. niger</i> carring sTF05 and the mCherry reporter (BBa_K4129123). The <i>A. niger</i> is grown on solid media plates. The plates contained either minimal media, minimal media with mM benzoic acid or MM with 0.6 g/L furfural.
-The fluorescence was assessed using the Vilber Fusion FX imager system to measure fluorescence shown as grey-white intensity. The exposure time was normalised to the fluorescences from genomic integrated BBa_K3046004, and this enabled comparison between plates. The exposure time used was 1.04 seconds.
+The fluorescence was assessed, after four days of incubation at 30<span>&#8451;</span>, using the Vilber Fusion FX imager system. A grey-white intensity scale was used to present the measured fluorescence. The exposure time was normalised to the fluorescences from genomic integrated BBa_K3046004, and this enabled comparison between plates. The exposure time used was 1.04 seconds.
 It is observed that genomic integrated BBa_K3046004 displays fluorescence and the negative control of BBa_K4129025 did not. It should be noted that no growth was observed on the plates containing 0.6 g/L furfural (figure 1).
@@ Line 24: / Line 24: @@
-The observed fluorescence of FunsTF05 was greater than that of genomic integrated BBa_K304600 on the different plates. The observed fluorescence of FunsTF05 did not differ too much between plates, which indicate FunsTF05 constitutively expressed mCherry (BBa_K4129123) (figure 2).
+The observed fluorescence of FunsTF05 was greater than that of genomic integrated BBa_K3046004 on the different plates (figure 2). BBa_K3046004 is a strong constitutive promoter in <i>A</i>. <i>niger</i>, and this emphasise the stregth of FunsTF05.
+ The observed fluorescence of FunsTF05 did not differ too much between plates, which indicate FunsTF05 constitutively expressed mCherry (BBa_K4129123) (figure 2).