Difference between revisions of "Part:BBa K4032104"
 
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<partinfo>BBa_K4032104 short</partinfo>
 
<partinfo>BBa_K4032104 short</partinfo>
  
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This part contains sequences of fuse protein of amylase and RFP.
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Contents :
  
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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.
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・The lac promoter and lacZ from <partinfo>BBa_J33207</partinfo>
  
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This enzyme hydrolyzes of (1-4)-alpha-D-glycosidic linkages in polysaccharides containing three or more (1-4)-alpha-linked D-glucose units.
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・The native RBS from <partinfo>BBa_K523001</partinfo>
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Red fluorescence of RFP is observed under the fluorescence microscope. 
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This part was created by In-Fusion method using BBa_J04450 as inverse and BBa_K523006 as insert.
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・The gene codes for the amylase-RFP fusion protein. (<partinfo>BBa_K4032003</partinfo>）
  
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・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].
  
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【plasmid image】
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===Usage and Biology===
  
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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].
  
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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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Fig. 1 method
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===Design===
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The lac promoter and the double terminator are added to <partinfo>BBa_K4032006</partinfo>, forming this part.
  
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The lac promoter and the double terminator are from <partinfo>BBa_J04450</partinfo>.
  
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Experiments
 
  
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【image】
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This part was created by In-Fusion method using <partinfo>BBa_J04450</partinfo> and <partinfo>BBa_K523006</partinfo> as an insert.
  
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Fig. 2　The growth of E.coli (DH5α) expressing of amylase･RFP
 
  
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Pre-culture was performed at 37°C for 9 hours. Incubation was carried out at 37°C, 130 rpm. Four hours after the start of incubation, 0.5 mM IPTG was added to the amylase･RFP culture solution when OD = 0.633. We measured OD600 every approximately 4 hours.
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https://2021.igem.org/wiki/images/thumb/1/12/T--Gunma--amylase-rfp-design.png/800px-T--Gunma--amylase-rfp-design.png
  
  
  
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Fig. 1　The plasmid design of BBa_K4032104
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==Experiments==
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===Time course===
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https://2021.igem.org/wiki/images/a/a1/T--Gunma--amylase-RFP-timecourse-dh5%CE%B1.png
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Fig. 2　The growth of ''E. coli'' (DH5α) expressing amylase-RFP fusion protein
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Pre-culture : 37 ℃,9 h (130 rpm)
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Culture : 37 ℃ (130 rpm)
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･4 hours after, adding 0.5 mM IPTG to the amylase-RFP (OD = 0.63)
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･Measuring OD600 every approx. 4 hours
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===SDS-PAGE===
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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.
  
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SDS
 
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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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【image】
 
  
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Figure ??. 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.
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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, supernatant.
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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.


800px-T--Gunma--amylase-rfp-design.png


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.


T--Gunma--amylase-RFP-SDS-PAGE.png
















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]