Difference between revisions of "Part:BBa K3611008"

Latest revision as of 15:22, 27 October 2020

hrp Bio-Amplifier

The natural hrp regulatory network contains activator protein HrpR and HrpS. These two would form an ultrasensitive high-order co-complex HrpRS, which bind the upstream activator sequence of the hrpL promoter to remodel the closed σ54-RNAP-hrpL transcription complex to an open one through ATP hydrolysis [1]. This have been proven to amplify the input transcriptional signal. We used promoter LacUV5 as the input of hrp amplifier with EGFP as the output, to verify the amplification rate of the amplifier (Fig. 1).

Fig. 1 The architecture of transduction signal amplifier based on hrp regulatory system. LacUV5 promoter, the gene downstream of this promoter will be transcribed under the IPTG activation. The hrpR and hrpS are activator protein’s coding genes, PhrpL, a promoter which can be induced by the ultrasensitive high-order co-complex HrpRS. The ribosome binding sites (RBS) B0030 and terminator B0015 were indicated as gray. The enhanced fluorescent protein (EGFP) was used as our reporter protein, with egfp as its coding gene.

We transformed 3 different vectors into E. coli BL21 (DE3) competent cells: the egfp expression vector V-egfp without amplifier, V-hrp-egfp with amplifier and the empty vector V-E. Then, we use LB-K (LB with Kanamycin) to culture bacteria. Once we got a single bacteria, 5ml LB-K liquid medium was used to cultivate a single colony at 37℃ overnight. After cultivation we wash the bacteria with 1ml PBS and measure the cell growth, by using UV spectrophotometer. Then we used LB-K to dilute bacteria and added them into transparent 96-well plate with OD600=0.05 per well. Four different concentration gradients of Isopropyl-beta-D-thiogalactopyranoside (IPTG)（0, 10-6, 10-5, 10-4, 10-3 mmol/L） were used to induce the expression of every group. After 1h IPTG induction, we used the Enzyme Labeling Instrument to detect the green fluorescent signal with the excitation wavelength at 485nm and the absorption wavelength at 525nm.

When the promoter is connected in series with the hrp amplifier, the output signal amplitude increased has a great improvement (Fig. 2).

Fig. 2 Responses of the EGFP without hrp transduction amplifier (V-egfp) and with amplifier (V-hrp-egfp). The cells are induced by 5 varying concentrates of IPTG (0, 10-6, 10-5, 10-4, 10-3 mmol/L) after 1h.

Reference

[1]Milija Jovanovic et al. "Regulation of the co-evolved HrpR and HrpS AAA+ proteins required for Pseudomonas syringae pathogenicity". 2.Pt 4(2011):567-573.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 1897
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 1879
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 1089
Illegal SapI.rc site found at 1722

@@ Line 9: / Line 9: @@
-Fig. 1 The architecture of transduction signal amplifier based on hrp regulatory system. LacUV5 promoter, the gene downstream of this promoter will be transcribed under the IPTG activation. The hrpR and hrpS are activator protein’s coding genes, PhrpL, a promoter which can be induced by the ultrasensitive high-order co-complex HrpRS. The ribosome binding sites (RBS) B0030 and terminator B0015 were indicated as gray. The enhanced fluorescent protein (EGFP) was used as our reporter protein, with egfp as its coding gene.
+'''Fig. 1 The architecture of transduction signal amplifier based on hrp regulatory system.''' LacUV5 promoter, the gene downstream of this promoter will be transcribed under the IPTG activation. The hrpR and hrpS are activator protein’s coding genes, PhrpL, a promoter which can be induced by the ultrasensitive high-order co-complex HrpRS. The ribosome binding sites (RBS) B0030 and terminator B0015 were indicated as gray. The enhanced fluorescent protein (EGFP) was used as our reporter protein, with egfp as its coding gene.
 We transformed 3 different vectors into E. coli BL21 (DE3) competent cells: the egfp expression vector V-egfp without amplifier, V-hrp-egfp with amplifier and the empty vector V-E. Then, we use LB-K (LB with Kanamycin) to culture bacteria. Once we got a single bacteria, 5ml LB-K liquid medium was used to cultivate a single colony at 37℃ overnight. After cultivation we wash the bacteria with 1ml PBS and measure the cell growth, by using UV spectrophotometer. Then we used LB-K to dilute bacteria and added them into transparent 96-well plate with OD600=0.05 per well. Four different concentration gradients of Isopropyl-beta-D-thiogalactopyranoside (IPTG)（0, 10-6, 10-5, 10-4, 10-3 mmol/L） were used to induce the expression of every group. After 1h IPTG induction, we used the Enzyme Labeling Instrument to detect the green fluorescent signal with the excitation wavelength at 485nm and the absorption wavelength at 525nm.
@@ Line 17: / Line 17: @@
 https://static.igem.org/mediawiki/parts/5/5c/T--NEU_CHINA--mp137.png
-Fig. 2 Responses of the EGFP without hrp transduction amplifier (V-egfp) and with amplifier (V-hrp-egfp). The cells are induced by 5 varying concentrates of IPTG (0, 10-6, 10-5, 10-4, 10-3 mmol/L) after 1h.
+'''Fig. 2 Responses of the EGFP without hrp transduction amplifier (V-egfp) and with amplifier (V-hrp-egfp).''' The cells are induced by 5 varying concentrates of IPTG (0, 10-6, 10-5, 10-4, 10-3 mmol/L) after 1h.
-Reference
+'''Reference'''
-[1]Jovanovic,M., James,E.H., Burrows,P.C., Rego,F.G.M., Buck,M. and Schumacher,J. (2011) Regulation of the co-evolved HrpR and HrpS AAA+ proteins required for Pseudomonas syringae pathogenicity. Nat. Commun., 2:177.
+[1]Milija Jovanovic et al. "Regulation of the co-evolved HrpR and HrpS AAA+ proteins required for Pseudomonas syringae pathogenicity". 2.Pt 4(2011):567-573.