Difference between revisions of "Part:BBa K4291009"

Latest revision as of 13:45, 12 October 2022

SLC7A5

Improvement of an existing part

Autism Spectrum Disorder (ASD) is a neuro-developmental disorder characterized by social communication disorders. Our research aims at early diagnosis and accurately diagnosing and treatment. In order to carry forward the spirit of iGEM, we specially searched the iGEM Biological Parts library for related projects and picked BBa_K302033, MazF. This is a biological part submitted by iGEM10_Newcastle in 2010, they developed an ASD diagnosis and treatment method, but didn’t totally achieve ASD treatment through the brain-gut-axis. So that it is really important to provide another candidate protein for ASD treatment. In this project, our team carried out a related protein SLC7A4 for this part in the laboratory. The SLC7A5 transports the so-called branched-chain amino acids (BCAA) into the brain. Mutation in this gene reduces branched-chain amino acid levels in the brain and interferes with neural cell protein synthesis which reduced social interactions and other changes. Improving the SLC7A5 levels by building engineered probiotics may achieve the treatment of autism. In order to verify if the new part SLC7A5 we developed worked well, we transformed the recombinant plasmid into E. coli BL21(DE3) to verify the protein expression level by SDS-PAGE. This protein may could be used in future research.

Profile

Name: SLC7A5

Base Pairs: 1524 bp

Origin: Homo sapiens, genome

Properties: amino acid transporter

Usage and biology

Solute carrier transporter 7a5 (SLC7A5), also known as LAT1, belongs to the APC (amino acid, polyamine, choline) superfamily. The heterodimer SLC7A5 and glycoprotein CD98 linked through a conserved disulfide is responsible for the uptake of essential amino acids in crucial body districts such as the placenta and blood-brain barrier. The SLC7A5 transports the so-called branched-chain amino acids (BCAA) into the brain. Mutation in this gene reduces branched-chain amino acid levels in the brain and interferes with neural cell protein synthesis which shows reduced social interactions and other changes. There are many different genetic mutations causing autism, and this heterogeneity makes it difficult to develop effective treatments.

Construct design

SLC7A5 transporter is expressed under the promoter T7. The gene fragment was inserted into pET28a.

Figure 1. The map of pET28a-SLC7A5

Experimental approach

1.1 Construction of biosensor expression plasmids

In order to build our plasmids, we let the synthetic company synthesize the SLC7A5 DNA sequence and inserted it into the pUC57 vector. We amplified the SLC7A5 from the plasmid by PCR (Figure 2) and inserted it into the EcoRI and XhoI sites of the pET28a vector. We transformed it into E. coli Top10 competent cells.

Figure 2. SLC7A5 amplification electrophoresis result.

1.2 Colony PCR of pET28a-SLC7A5

We selected the 8 colonies to perform by colony-PCR (Figure 3). The electrophoresis result showed that No. 1, 2, and 7 has correct bands.

Figure 3. colony PCR electrophoresis result.

1.2 Sanger sequencing of recombinant plasmid

We inoculated the correct colonies in LB (Kan+) liquid medium and extracted the plasmids and sent them to the company for Sanger sequencing. The returned sequencing data showed that there were no mutations in the gene region which means we successfully constructed the recombinant plasmid.

Figure 4. Sanger sequencing.

Proof of function

SLC7A5 purification

In order to verify if SLC7A5 expression in the E. coli host strain, we transformed the recombinant plasmids into E. coli BL21(DE3), inoculated the recombinants and added IPTG to induce protein expression when the OD600 reached 0.6. After overnight induce and culture, we collected the cells and ultrasonic fragmentation of cells to release the intracellular proteins. Next, we verified the expression level of the target protein by SDS-PAGE (Figure 5). As a result, the recombinants successfully expressed our target protein.

Figure 5. SDS-page of SLC7A5 purification.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 30
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 39
Illegal NgoMIV site found at 108
1000
COMPATIBLE WITH RFC[1000]

@@ Line 4: / Line 4: @@
 SLC7A5
-== Characterization by Canton_HS ==
-== BBa_K4291009 ==
+==Improvement of an existing part==
+Autism Spectrum Disorder (ASD) is a neuro-developmental disorder characterized by social communication disorders. Our research aims at early diagnosis and accurately diagnosing and treatment.
+In order to carry forward the spirit of iGEM, we specially searched the iGEM Biological Parts library for related projects and picked BBa_K302033, MazF. This is a biological part submitted by iGEM10_Newcastle in 2010, they developed an ASD diagnosis and treatment method, but didn’t totally achieve ASD treatment through the brain-gut-axis. So that it is really important to provide another candidate protein for ASD treatment.
+In this project, our team carried out a related protein SLC7A4 for this part in the laboratory. The SLC7A5 transports the so-called branched-chain amino acids (BCAA) into the brain. Mutation in this gene reduces branched-chain amino acid levels in the brain and interferes with neural cell protein synthesis which reduced social interactions and other changes. Improving the SLC7A5 levels by building engineered probiotics may achieve the treatment of autism.
+In order to verify if the new part SLC7A5 we developed worked well, we transformed the recombinant plasmid into E. coli BL21(DE3) to verify the protein expression level by SDS-PAGE. This protein may could be used in future research.
+== Profile ==
 Name: SLC7A5
 Base Pairs: 1524 bp
 Origin: Homo sapiens, genome
 Properties: amino acid transporter
 == Usage and biology ==
 Solute carrier transporter 7a5 (SLC7A5), also known as LAT1, belongs to the APC (amino acid, polyamine, choline) superfamily. The heterodimer SLC7A5 and glycoprotein CD98 linked through a conserved disulfide is responsible for the uptake of essential amino acids in crucial body districts such as the placenta and blood-brain barrier. The SLC7A5 transports the so-called branched-chain amino acids (BCAA) into the brain. Mutation in this gene reduces branched-chain amino acid levels in the brain and interferes with neural cell protein synthesis which shows reduced social interactions and other changes. There are many different genetic mutations causing autism, and this heterogeneity makes it difficult to develop effective treatments.
-== Construct design =
+== Construct design ==
 SLC7A5 transporter is expressed under the promoter T7. The gene fragment was inserted into pET28a.
 [[File:T--Canton HS--BBa K4291009-figure 11.jpg|500px|thumb|center|Figure 1. The map of pET28a-SLC7A5]]
@@ Line 18: / Line 29: @@
 == Experimental approach ==
 .1 Construction of biosensor expression plasmids
-In order to build our plasmids, we let the synthetic company synthesize the SLC7A5 DNA sequence and inserted it into the pUC57 vector. We amplified the SLC7A5 from the plasmid by PCR (Figure 1A) and inserted it into the EcoRI and XhoI sites of the pET28a vector. We transformed it into E. coli Top10 competent cells.
+In order to build our plasmids, we let the synthetic company synthesize the SLC7A5 DNA sequence and inserted it into the pUC57 vector. We amplified the SLC7A5 from the plasmid by PCR (Figure 2) and inserted it into the EcoRI and XhoI sites of the pET28a vector. We transformed it into E. coli Top10 competent cells.
 [[File:T--Canton HS--BBa K4291009-figure 2.jpg|500px|thumb|center|Figure 2. SLC7A5 amplification electrophoresis result.]]
 .2 Colony PCR of pET28a-SLC7A5
 We selected the 8 colonies to perform by colony-PCR (Figure 3). The electrophoresis result showed that No. 1, 2, and 7 has correct bands.
 [[File:T--Canton HS--BBa K4291009-figure 3.png|500px|thumb|center|Figure 3. colony PCR electrophoresis result.]]
+.2 Sanger sequencing of recombinant plasmid
+We inoculated the correct colonies in LB (Kan+) liquid medium and extracted the plasmids and sent them to the company for Sanger sequencing. The returned sequencing data showed that there were no mutations in the gene region which means we successfully constructed the recombinant plasmid.
+[[File:T--Canton HS--BBa K4291009-figure 4.jpg|500px|thumb|center|Figure 4. Sanger sequencing.]]
+== Proof of function ==
+SLC7A5 purification
+In order to verify if SLC7A5 expression in the E. coli host strain, we transformed the recombinant plasmids into E. coli BL21(DE3), inoculated the recombinants and added IPTG to induce protein expression when the OD600 reached 0.6. After overnight induce and culture, we collected the cells and ultrasonic fragmentation of cells to release the intracellular proteins. Next, we verified the expression level of the target protein by SDS-PAGE (Figure 5). As a result, the recombinants successfully expressed our target protein.
+[[File:T--Canton HS--BBa K4291009-figure 55.jpg|500px|thumb|center|Figure 5. SDS-page of SLC7A5 purification.]]