Difference between revisions of "Part:BBa K4032104"
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Contents : | Contents : | ||
| − | ・The lac promoter and lacZ | + | ・The lac promoter and lacZ from <partinfo>BBa_J33207</partinfo> |
| − | ・The native RBS | + | ・The native RBS from <partinfo>BBa_K523001</partinfo> |
| − | ・The gene codes for the amylase-RFP fusion protein. | + | ・The gene codes for the amylase-RFP fusion protein. (<partinfo>BBa_K4032003</partinfo>） |
| − | ・The double terminator | + | ・The double terminator from <partinfo>BBa_B0015</partinfo> |
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| + | For more information on the amylase gene ''malS'',see [https://www.ncbi.nlm.nih.gov/gene/948088 NCBI: NC_000913.3]. | ||
===Usage and Biology=== | ===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. See [https://www.ncbi.nlm.nih.gov/gene/948088 NCBI: NC_000913.3] | + | 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. | 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. | ||
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Pre-culture : 37 ℃,9 h (130 rpm) | Pre-culture : 37 ℃,9 h (130 rpm) | ||
| − | Culture : 37 ℃ ( | + | Culture : 37 ℃ (130 rpm) |
･4 hours after, adding 0.5 mM IPTG to the amylase-RFP (OD = 0.63) | ･4 hours after, adding 0.5 mM IPTG to the amylase-RFP (OD = 0.63) | ||
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===SDS-PAGE=== | ===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 | + | 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. | The cultured bacteria were sonicated using Sonication buffer (Phosphate buffer solution (pH 7.0) + 150 mM NaCl + 10% glycerol) and SDS-PAGE was performed. | ||
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| − | + | [[image:T--Gunma--amylase-RFP-SDS-PAGE.png|400px|thumb|left]] | |
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| − | 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, | + | 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. |
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
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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1841
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 607
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE 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 - 1000COMPATIBLE WITH RFC[1000]