Difference between revisions of "Part:BBa K4032104"
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__NOTOC__ | __NOTOC__ | ||
<partinfo>BBa_K4032104 short</partinfo> | <partinfo>BBa_K4032104 short</partinfo> | ||
| − | |||
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Contents : | Contents : | ||
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・The lac promoter and lacZ (from <partinfo>BBa_J33207</partinfo>) | ・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.（<partinfo>BBa_K4032003</partinfo>） | ・The gene codes for the amylase-RFP fusion protein.（<partinfo>BBa_K4032003</partinfo>） | ||
・The double terminator (from <partinfo>BBa_B0015</partinfo>) | ・The double terminator (from <partinfo>BBa_B0015</partinfo>) | ||
| + | |||
| + | See [https://www.ncbi.nlm.nih.gov/gene/948088 NCBI: NC_000913.3]for information on the amylase gene ''malS''. | ||
===Usage and Biology=== | ===Usage and Biology=== | ||
| − | This enzyme hydrolyzes of (1-4)- | + | 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]for details. |
| − | In the case of this part, Red red fluorescence of RFP is observed | + | 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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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 added to <partinfo>BBa_K4032006</partinfo>, forming this part. | ||
| − | The lac promoter and the double terminator are | + | The lac promoter and the double terminator are from <partinfo>BBa_J04450</partinfo>. |
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| − | Fig. 2 The growth of E.coli (DH5α) expressing | + | Fig. 2 The growth of ''E. coli'' (DH5α) expressing amylase-RFP fusion protein |
| − | Pre-culture : 37 ℃, 9 h (130 rpm) | + | Pre-culture : 37 ℃,9 h (130 rpm) |
Culture : 37 ℃ (130rpm) | Culture : 37 ℃ (130rpm) | ||
| − | ･4 hours after, adding 0.5 mM IPTG to the | + | ･4 hours after, adding 0.5 mM IPTG to the amylase-RFP (OD = 0.63) |
･Measuring OD600 every approx. 4 hours | ･Measuring OD600 every approx. 4 hours | ||
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===SDS-PAGE=== | ===SDS-PAGE=== | ||
| − | To investigate the expression of | + | 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°C 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°C 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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| − | 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, solubility. | + | 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, solubility. |
Revision as of 06:58, 21 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)
See NCBI: NC_000913.3for information on the amylase gene malS.
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 NCBI: NC_000913.3for details.
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 ℃ (130rpm)
･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°C 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°C 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, solubility.
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]