Difference between revisions of "Part:BBa K4032104"

Latest revision as of 02:46, 22 October 2021

lacI+lacZ+RBS+amylase-RFP+double terminator

Contents :

・The lac promoter and lacZ from BBa_J33207

・The native RBS from BBa_K523001

・The gene codes for the amylase-RFP fusion protein. (BBa_K4032003）

・The double terminator from BBa_B0015

For more information on the amylase gene malS,see NCBI: NC_000913.3.

Usage and Biology

This enzyme hydrolyzes of (1-4)-α-D-glycosidic linkages in polysaccharides containing three or more (1-4)-α-linked D-glucose units. For more information, See NCBI: NC_000913.3.

In the case of this part, Red red fluorescence of RFP is observed with a fluorescence microscope, but isn’t observed under the natural light source.

Design

The lac promoter and the double terminator are added to BBa_K4032006, forming this part.

The lac promoter and the double terminator are from BBa_J04450.

This part was created by In-Fusion method using BBa_J04450 and BBa_K523006 as an insert.

Fig. 1　The plasmid design of BBa_K4032104

Experiments

Time course

T--Gunma--amylase-RFP-timecourse-dh5%CE%B1.png

Fig. 2　The growth of E. coli (DH5α) expressing amylase-RFP fusion protein

Pre-culture : 37 ℃,9 h (130 rpm)

Culture : 37 ℃ (130 rpm)

･4 hours after, adding 0.5 mM IPTG to the amylase-RFP (OD = 0.63)

･Measuring OD600 every approx. 4 hours

SDS-PAGE

To investigate the expression of amylase-RFP from E. coli and coral, bacteria transformed with BL21(DE3) were cultured in an LB medium containing chloramphenicol. After incubation of E. coli at 37 ℃ and 130 rpm for 16 hours, the cells were inoculated into a new medium and cultured in liquid until the logarithmic growth phase. IPTG was added to a final concentration of 0.2 mM, and E. coli was cultured overnight for 10 hours at 25 ℃ and 130 rpm.

The cultured bacteria were sonicated using Sonication buffer (Phosphate buffer solution (pH 7.0) + 150 mM NaCl + 10% glycerol) and SDS-PAGE was performed.

Fig. 3 SDS-PAGE gel for quantification of amylase-RFP. M, molecular mass markers; WT, wild type; amy-RFP, amylase-RFP from E. coli and coral; P, pellet; S, supernatant.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 1841
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 607
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 977
Illegal AgeI site found at 2142
Illegal AgeI site found at 3221
Illegal AgeI site found at 3333
1000
COMPATIBLE WITH RFC[1000]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K4032104 short</partinfo>
-This part contains sequences of fuse protein of amylase and RFP.
+Contents :
-This part is the addition of a lac promoter and double terminator to BBa_K40320xx. Lac promoter and double terminator were used as contained in BBa_J04450.
+・The lac promoter and lacZ from <partinfo>BBa_J33207</partinfo>
-This enzyme hydrolyzes of (1-4)-alpha-D-glycosidic linkages in polysaccharides containing three or more (1-4)-alpha-linked D-glucose units.
+・The native RBS from <partinfo>BBa_K523001</partinfo>
-Red fluorescence of RFP is observed under the fluorescence microscope.
-This part was created by In-Fusion method using BBa_J04450 as inverse and BBa_K523006 as insert.
+・The gene codes for the amylase-RFP fusion protein. (<partinfo>BBa_K4032003</partinfo>）
---------------------------------------
+・The double terminator from <partinfo>BBa_B0015</partinfo>
-plasmid image
+For more information on the amylase gene ''malS'',see [https://www.ncbi.nlm.nih.gov/gene/948088 NCBI: NC_000913.3].
+===Usage and Biology===
-------------------------------------
+This enzyme hydrolyzes of (1-4)-α-D-glycosidic linkages in polysaccharides containing three or more (1-4)-α-linked D-glucose units. For more information, See [https://www.ncbi.nlm.nih.gov/gene/948088 NCBI: NC_000913.3].
+In the case of this part, Red red fluorescence of RFP is observed with a fluorescence microscope, but isn’t observed under the natural light source.
+===Design===
+The lac promoter and the double terminator are added to <partinfo>BBa_K4032006</partinfo>, forming this part.
+The lac promoter and the double terminator are from <partinfo>BBa_J04450</partinfo>.
+This part was created by In-Fusion method using <partinfo>BBa_J04450</partinfo> and <partinfo>BBa_K523006</partinfo> as an insert.
+https://2021.igem.org/wiki/images/thumb/1/12/T--Gunma--amylase-rfp-design.png/800px-T--Gunma--amylase-rfp-design.png
+Fig. 1　The plasmid design of BBa_K4032104
+==Experiments==
+===Time course===
+https://2021.igem.org/wiki/images/a/a1/T--Gunma--amylase-RFP-timecourse-dh5%CE%B1.png
+Fig. 2　The growth of ''E. coli'' (DH5α) expressing amylase-RFP fusion protein
+Pre-culture : 37 ℃,9 h (130 rpm)
+Culture : 37 ℃ (130 rpm)
+･4 hours after, adding 0.5 mM IPTG to the amylase-RFP (OD = 0.63)
+･Measuring OD600 every approx. 4 hours
+===SDS-PAGE===
+To investigate the expression of amylase-RFP from ''E. coli'' and coral, bacteria transformed with BL21(DE3) were cultured in an LB medium containing chloramphenicol. After incubation of ''E. coli'' at 37 ℃ and 130 rpm for 16 hours, the cells were inoculated into a new medium and cultured in liquid until the logarithmic growth phase. IPTG was added to a final concentration of 0.2 mM, and E. coli was cultured overnight for 10 hours at 25 ℃ and 130 rpm.
+The cultured bacteria were sonicated using Sonication buffer (Phosphate buffer solution (pH 7.0) + 150 mM NaCl + 10% glycerol) and SDS-PAGE was performed.
+[[image:T--Gunma--amylase-RFP-SDS-PAGE.png|400px|thumb|left]]
+Fig. 3 SDS-PAGE gel for quantification of amylase-RFP. M, molecular mass markers; WT, wild type; amy-RFP, amylase-RFP from ''E. coli'' and coral; P, pellet; S, supernatant.
-Fig. 1 method