Difference between revisions of "Part:BBa K3580200"

Latest revision as of 11:51, 27 October 2020

Alanine racemase with 6xHis-Tag

BBa_K3580200 is an AR sequence with 6xHis tag for affinity purification method widely established with minimal impact on the activity and structure of the target protein.

This part includes the same sequence of alr as BBa_K1172901, but it is new having 6xHis sequence. 6xHis has the same amino acid sequence with BBa_K1223006 but some codons are different. We expressed alanine racemase from this parts with T5.

Background
Racemase catalyzes interconversion between L-amino acid and D-amino acid. There are many kinds of racemases and each corresponds to respective amino acid. Alanine racemase (AR) is a fold type III racemase enzyme catalyze the conversion of L-alanine to D-alanine [Walish 1989].This enzyme provides D-alanine consuming L-alanine and using pyridoxal 5- phosphate (PLP) as a cofactor. AR is unique to prokaryotes and plays an important role in a biosynthesis[Walish 1989] [Azam 2016]. Since D-alanine is used as a necessary component of the peptidoglycan layer of bacterial cell walls, lack of alanine racemase can result in the inhibition of growth of prokaryotes[Azam 2016]. As it is generally absent in higher eukaryotes such as human but is ubiquitous throughout bacteria. Therefore, alanine racemase is recognized as an attractive target for antibacterial drug development [Azam 2016].

Characterizations of the alanine racemase with 6xHis tag
Alanine racemase (AR) is a fold type III racemase enzyme catalyze the conversion of L-alanine to D-alanine [Walish 1989].This enzyme provides D-alanine consuming L-alanine and using pyridoxal 5- phosphate (PLP) as a cofactor(Fig.1).

Fig.1Catalysis of racemization of alanine by alanine racemase

BBa_K3580200 is an AR sequence with 6xHis tag. This part includes the same sequence of AR as BBa_K1172901, but it is new having 6xHis sequence. 6xHis has the same amino acid sequence with BBa_K1223006 at the 5’ end but some codons are different. This 6x His-tag facilitates the purification of alanine racemase by affinity purification. This part’s sequence does not include BioBrick(RFC 10) sequences. In our project, we inserted this part between bacteriophage T5 promoter for E. coli RNA polymerase and lambda t0 terminator of pCA24N(-gfp) planmid. The sequence of this promoter is partly consistent with part BBa_K592008, T5-lac Promoter. This allows for the induction of expression by IPTG.

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.2). The meanings of each abbreviation are given in Table 1. The concentration of purified racemase was quantified with the Bradford method.

Fig.2 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.3).

Fig.3 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 

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 367
Illegal NotI site found at 1121
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 291
Illegal BamHI site found at 21
Illegal BamHI site found at 993
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 409
Illegal AgeI site found at 709
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 166

@@ Line 9: / Line 9: @@
 We expressed alanine racemase from this parts with T5.
-Expression and purification of alanine racemase
+<b>Background</b><br>
+Racemase catalyzes interconversion between L-amino acid and D-amino acid. There are many kinds of racemases and each corresponds to respective amino acid. Alanine racemase (AR) is a fold type III racemase enzyme catalyze the conversion of L-alanine to D-alanine [Walish 1989].This enzyme provides D-alanine consuming L-alanine and using pyridoxal 5- phosphate (PLP) as a cofactor. AR is unique to prokaryotes and plays an important role in a biosynthesis[Walish 1989] [Azam 2016]. Since D-alanine is used as a necessary component of the peptidoglycan layer of bacterial cell walls, lack of alanine racemase can result in the inhibition of growth of prokaryotes[Azam 2016]. As it is generally absent in higher eukaryotes such as human but is ubiquitous throughout bacteria. Therefore, alanine racemase is recognized as an attractive target for antibacterial drug development [Azam 2016].
-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.
+<b>Characterizations of the alanine racemase with 6xHis tag</b><br>
+Alanine racemase (AR) is a fold type III racemase enzyme catalyze the conversion of L-alanine to D-alanine [Walish 1989].This enzyme provides D-alanine consuming L-alanine and using pyridoxal 5- phosphate (PLP) as a cofactor(Fig.1).
-[[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]]
+[[File:T--Waseda--racemase_parts_racemization.png|500px|thumb|center|Fig.1Catalysis of racemization of alanine by alanine racemase]]
+BBa_K3580200 is an AR sequence with 6xHis tag. This part includes the same sequence of AR as BBa_K1172901, but it is new having 6xHis sequence. 6xHis has the same amino acid sequence with BBa_K1223006 at the 5’ end but some codons are different. This 6x His-tag facilitates the purification of alanine racemase by affinity purification. This part’s sequence does not include BioBrick(RFC 10) sequences.
+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. The sequence of this promoter is partly consistent with part BBa_K592008, T5-lac Promoter. This allows for the induction of expression by IPTG.
+<b>Expression and purification of alanine racemase</b><br>
+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.2). The meanings of each abbreviation are given in Table 1. The concentration of purified racemase was quantified with the Bradford method.
+[[File:T--Waseda--racemase_parts_purification_sds.png|500px|thumb|center|Fig.2 SDS-PAGEs of purified <i>E. coli</i> BL21(DE3)star containing BBa_K3580200]]
 [[File:T--Waseda--racemase_parts_table1.png|400px|thumb|center|Table.1 Each meaning of abbriviation ]]
@@ Line 19: / Line 31: @@
 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).
+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.3).
-[[File:T--Waseda--racemase_parts_201023_bar.png|400px|thumb|center|  Fig.2 Effect of L-alanine substrate repletion for translation by racemase ]]
+[[File:T--Waseda--racemase_parts_201023_bar.png|400px|thumb|center|  Fig.3 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.