Difference between revisions of "Part:BBa K3580200:Experience"

 
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===Applications of BBa_K3580200===
 
===Applications of BBa_K3580200===
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Expression and purification of alanine racemase
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In our project, we inserted this part between bacteriophage T5 promoter for <i>E. coli</i> RNA polymerase and lambda t0 terminator of pCA24N(-gfp) planmid. This plasmid was transformed into the BL21(DE3) star strain. As this vector is chloramphenicol resistant, we always cultured the bacteria in media containing chloramphenicol. After expression induction by IPTG, the BL21 cells were collected by centrifugation, then sonicated and His-Tag purification was performed. (wikiマテメソハイパーリンク) The His-Tag purified Alr was confirmed with SDS-PAGE(Fig.1). The meanings of each abbreviation are given in Table 1. The concentration of purified racemase was quantified with the Bradford method.
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[[File:T--Waseda--racemase_parts_purification_sds.png|500px|thumb|center|Fig.1 SDS-PAGEs of purified <i>E. coli</i> BL21(DE3)star containing BBa_K3580200]]
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[[File:T--Waseda--racemase_parts_table1.png|400px|thumb|center|Table.1 Each meaning of abbriviation ]]
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We decided to assay AR by using PURE frex provided by GeneFrontier, Inc. As PURE frex does not contain any extra metabolic enzymes other than those involved in transcription and translation, we would be able to perform the assays without considering the problems of controlling the metabolic system. (wikiマテメソハイパーリンク) 
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Consequently, to assay the activity of purified AR, the racemization reaction was performed with the following solution(Table.2) and incubated at 37°C for 1 h. The sample was Filtered using centrifugal filter device at 14,000g for 30min (Amicon Ultra-0.5 Centrifugal Filter Devices 3K) and the filtered sample was collected. The filter device was washed prior to centrifugation of the sample using the buffer used in the AR reaction. the sample was centrifuged to collect the filtered product. Filtering allowed us to collect substances other than AR from the incubated product. Fluorescence values of GFPS1 synthesized by the combination of PURE frex containing 19 L-amino acids and a plasmid encoding the reporter protein GFPS1 with L-alanine, D-alanine, and the reaction solution filtration products of AR and D-alanine (presumably containing PLP, potassium phosphate, and racemic alanine) were measured(Fig.2).
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[[File:T--Waseda--racemase_parts_201023_bar.png|400px|thumb|center|  Fig.2 Effect of L-alanine substrate repletion for translation by racemase ]]
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The results demonstrated that GFP could not be synthesized by D-alanine alone, but L-alanine produced by racemase-mediated racemization of D-alanine and L-alanine could be used for translation, creating a situation in which fluorescence was restored by the synthesized GFP.  
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[[File:T--Waseda--racemase_parts_table2.png |400px|thumb|center|Table.2  Alr racemization reaction assay solution  ]]
  
 
===User Reviews===
 
===User Reviews===

Latest revision as of 07:57, 27 October 2020


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Applications of BBa_K3580200

Expression and purification of alanine racemase

In our project, we inserted this part between bacteriophage T5 promoter for E. coli RNA polymerase and lambda t0 terminator of pCA24N(-gfp) planmid. This plasmid was transformed into the BL21(DE3) star strain. As this vector is chloramphenicol resistant, we always cultured the bacteria in media containing chloramphenicol. After expression induction by IPTG, the BL21 cells were collected by centrifugation, then sonicated and His-Tag purification was performed. (wikiマテメソハイパーリンク) The His-Tag purified Alr was confirmed with SDS-PAGE(Fig.1). The meanings of each abbreviation are given in Table 1. The concentration of purified racemase was quantified with the Bradford method.

Fig.1 SDS-PAGEs of purified E. coli BL21(DE3)star containing BBa_K3580200
Table.1 Each meaning of abbriviation

We decided to assay AR by using PURE frex provided by GeneFrontier, Inc. As PURE frex does not contain any extra metabolic enzymes other than those involved in transcription and translation, we would be able to perform the assays without considering the problems of controlling the metabolic system. (wikiマテメソハイパーリンク) 

Consequently, to assay the activity of purified AR, the racemization reaction was performed with the following solution(Table.2) and incubated at 37°C for 1 h. The sample was Filtered using centrifugal filter device at 14,000g for 30min (Amicon Ultra-0.5 Centrifugal Filter Devices 3K) and the filtered sample was collected. The filter device was washed prior to centrifugation of the sample using the buffer used in the AR reaction. the sample was centrifuged to collect the filtered product. Filtering allowed us to collect substances other than AR from the incubated product. Fluorescence values of GFPS1 synthesized by the combination of PURE frex containing 19 L-amino acids and a plasmid encoding the reporter protein GFPS1 with L-alanine, D-alanine, and the reaction solution filtration products of AR and D-alanine (presumably containing PLP, potassium phosphate, and racemic alanine) were measured(Fig.2).

Fig.2 Effect of L-alanine substrate repletion for translation by racemase

The results demonstrated that GFP could not be synthesized by D-alanine alone, but L-alanine produced by racemase-mediated racemization of D-alanine and L-alanine could be used for translation, creating a situation in which fluorescence was restored by the synthesized GFP.  

Table.2  Alr racemization reaction assay solution 

User Reviews

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