Difference between revisions of "Part:BBa K4477011"

 
 
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<partinfo>BBa_K4477011 short</partinfo>
 
<partinfo>BBa_K4477011 short</partinfo>
  
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This sequence codes for a full IgG anti-apoB(MDA). It is derived partially from Orticumab, a human antibody that is currently in development.
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===Overview===
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This sequence codes for a hybrid human/mouse anti-apoB(MDA) IgG antibody. It is derived partially from orticumab, a human antibody that is currently in development as a therapeutic drug. ApoB(MDA) is a specific chemical structure found as a component of oxidized low density lipoprotein (oxLDL). OxLDL is a biomarker of many inflammatory diseases such as atherosclerosis, so antibodies able to target oxLDL could enable potential detection and therapeutic applications for such diseases.  
  
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Past researchers have found success in expressing full-length antibodies in SHuffle when the heavy and light chains are arranged polycistronically; that is, they are transcribed onto the same mRNA, and then translated into separate proteins downstream [Nature paper]. Thus, this composite part includes just one promoter and one terminator, which cause both the coding sequence (CDS) for the light chain and that for the heavy chain to be transcribed together. The promoter is [part number], a strong T7 promoter that allows this composite part to be expressed through IPTG induction. The terminator is [part number], a two-way terminator that not only prevents RNA polymerases from reading past the composite part into the backbone, but prevents any genes in the backbone from being transcribed into the composite part. Bracketed by the promoter and terminator are the two translational units: first, the RBS and CDS for the light chain, and then the RBS and CDS for the heavy chain. Both RBSs were designed using the Salis RBS calculator to promote optimal translation of their respective CDSs and can be found under [name of/links to these basic parts].  
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Past researchers have found success in expressing full-length antibodies in SHuffle when the heavy and light chains are arranged polycistronically; that is, they are transcribed onto the same mRNA, and then translated into separate proteins downstream (1). Thus, this composite part includes just one promoter and one terminator, which cause both the coding sequence (CDS) for the light chain and that for the heavy chain to be transcribed together. The promoter is BBa_I712074, a strong T7 promoter that allows this composite part to be expressed through IPTG induction. The terminator is BBa_B0014, a two-way terminator that not only prevents RNA polymerases from reading past the composite part into the backbone, but prevents any genes in the backbone from being transcribed into the composite part. Bracketed by the promoter and terminator are the two translational units: first, the RBS and CDS for the light chain, and then the RBS and CDS for the heavy chain. Both RBSs were designed using the Salis RBS calculator to promote optimal translation of their respective CDSs and can be found in the Registry as parts BBa_K4477006 and BBa_K4477006.  
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Both the light and heavy chains of a full-length IgG contain multiple subunits, as shown in the diagram below [will make a diagram]. The light chain CDS encodes the light chain subunits in the following order, from upstream to downstream: variable light (VL), constant light (CL). The entire light chain amino acid sequence is identical to that of Orticumab, although codons were optimized for E. coli and to domesticate the sequence for RFC[10] and Type IIS assembly methods. The heavy chain CDS encodes the following subunits, from upstream to downstream: variable heavy (VH), constant heavy 1 (CH1), constant heavy 2 (CH2), constant heavy 3 (CH3). The VH and CH1 amino acid sequences were derived from Orticumab, while the CH2 and CH3 domains were derived from a murine (house mouse; Mus musculus) IgG heavy chain. Note that legal restriction enzyme sites were included between each subcomponent for modularity. Using these restriction enzymes, one can easily switch out the RBS to increase or decrease translational rates, for instance.
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Both the light and heavy chains of a full-length IgG contain multiple subunits, as shown in the diagram below.
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[[File:IgG_Subunits.png|600px|thumb|center|Figure 1. IgG Subunits. Diagram used under Creative Commons license.]]
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The light chain CDS encodes the light chain subunits in the following order, from upstream to downstream: variable light (VL), constant light (CL). The entire light chain amino acid sequence is identical to that of orticumab, although codons were optimized for <em>E. coli</em> and to domesticate the sequence for RFC[10] and Type IIS assembly methods. The heavy chain CDS encodes the following subunits, from upstream to downstream: variable heavy (VH), constant heavy 1 (CH1), constant heavy 2 (CH2), constant heavy 3 (CH3). The VH and CH1 amino acid sequences were derived from orticumab, while the CH2 and CH3 domains were derived from a murine (house mouse; <em>Mus musculus</em>) IgG heavy chain. Note that legal restriction enzyme sites were included between each subcomponent for modularity. Using these restriction enzymes, one can easily switch out the RBS to increase or decrease translational rates, for instance.
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[[File:anti-oxldl_igg_plasmid.png|600px|thumb|center|Figure 2. Plasmid map.]]
  
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<!-- Add more about the biology of this part here
 
 
===Usage and Biology===
 
===Usage and Biology===
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Orticumab was developed by Abcentra, a biopharmaceutical company that specializes in developing therapeutic antibodies targeting oxLDL with the overarching goal of treating cardiovascular inflammation.
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Orticumab is a fully-human monoclonal antibody that binds to oxLDL.  A proof-of-activity phase 2 trial - clinical trial NCT04776629 - is currently being conducted by Abcentra to test the safety and activity of the antibody in patients with severe psoriasis and cardiometabolic risk factors (2).  Pre-clinical research of orticumab suggests the possibility of the antibody to inhibit lipoprotein-a (Lp(a)) formation, reduce atherosclerosis, and reduce the thickening of blood vessels.
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Orticumab is currently being tested for its ability to reduce atherosclerosis and the thickening of blood vessels.  In rodent models, testing has suggested orticumab can reduce the progression of atherosclerosis, regress existing atherosclerotic lesions, and stabilize plaque.  In obese rehesus monkeys, orticumab has shown to reduce periperal inflammatory biomarkers (3).
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===Characterization===
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====BioBricking====
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We successfully BioBricked our human/mouse anti-oxLDL IgG-encoding device.
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[[File:anti-oxldl_igg_ge.png|600px|thumb|center|Figure 3. Restriction Digest Verification of Anti-oxLDL Plasmid Construct. (A) Virtual digests with BamHI and HindIII on Benchling yield one and two bands at 4.9 kb and 3 kb and 1.9 kb respectively. (B) 1: Anti-oxLDL colony uncut, 1 band below 4.9 kb. 3: Anti-oxLDL cut with HindIII, 3kb, 1.9 kb. 6: Anti-oxLDL cut with BamHI, 1 band at 4.9 kb.]]
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After transformation of the pSB3K3+Labeled anti-oxLDL ligation product into DH5-alpha, plasmids were extracted from 6 white colonies (expected to contain the plasmid of interest) and 1 pink colony (not expected to contain the plasmid of interest) for digestion analysis. In Figure 3, uncut colonies (such as that in lane 1) showed one band lower on the gel than the molecular weight we would expect to see when the plasmid is linearized, indicating the plasmids are high quality and not contaminated with restriction enzymes. Colonies cut with HindIII show two bands at 3 kb and 1.9 kb, as predicted in the virtual digest. Colonies cut with BamHI show one band just under 5 kb, also as predicted.
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K4477011 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4477011 SequenceAndFeatures</partinfo>

Latest revision as of 02:32, 14 October 2022


Human/mouse anti-apoB(MDA) (anti-oxLDL) IgG - complete expression cassette

Overview

This sequence codes for a hybrid human/mouse anti-apoB(MDA) IgG antibody. It is derived partially from orticumab, a human antibody that is currently in development as a therapeutic drug. ApoB(MDA) is a specific chemical structure found as a component of oxidized low density lipoprotein (oxLDL). OxLDL is a biomarker of many inflammatory diseases such as atherosclerosis, so antibodies able to target oxLDL could enable potential detection and therapeutic applications for such diseases.

Past researchers have found success in expressing full-length antibodies in SHuffle when the heavy and light chains are arranged polycistronically; that is, they are transcribed onto the same mRNA, and then translated into separate proteins downstream (1). Thus, this composite part includes just one promoter and one terminator, which cause both the coding sequence (CDS) for the light chain and that for the heavy chain to be transcribed together. The promoter is BBa_I712074, a strong T7 promoter that allows this composite part to be expressed through IPTG induction. The terminator is BBa_B0014, a two-way terminator that not only prevents RNA polymerases from reading past the composite part into the backbone, but prevents any genes in the backbone from being transcribed into the composite part. Bracketed by the promoter and terminator are the two translational units: first, the RBS and CDS for the light chain, and then the RBS and CDS for the heavy chain. Both RBSs were designed using the Salis RBS calculator to promote optimal translation of their respective CDSs and can be found in the Registry as parts BBa_K4477006 and BBa_K4477006.

Both the light and heavy chains of a full-length IgG contain multiple subunits, as shown in the diagram below.

Figure 1. IgG Subunits. Diagram used under Creative Commons license.

The light chain CDS encodes the light chain subunits in the following order, from upstream to downstream: variable light (VL), constant light (CL). The entire light chain amino acid sequence is identical to that of orticumab, although codons were optimized for E. coli and to domesticate the sequence for RFC[10] and Type IIS assembly methods. The heavy chain CDS encodes the following subunits, from upstream to downstream: variable heavy (VH), constant heavy 1 (CH1), constant heavy 2 (CH2), constant heavy 3 (CH3). The VH and CH1 amino acid sequences were derived from orticumab, while the CH2 and CH3 domains were derived from a murine (house mouse; Mus musculus) IgG heavy chain. Note that legal restriction enzyme sites were included between each subcomponent for modularity. Using these restriction enzymes, one can easily switch out the RBS to increase or decrease translational rates, for instance.

Figure 2. Plasmid map.

Usage and Biology

Orticumab was developed by Abcentra, a biopharmaceutical company that specializes in developing therapeutic antibodies targeting oxLDL with the overarching goal of treating cardiovascular inflammation.

Orticumab is a fully-human monoclonal antibody that binds to oxLDL. A proof-of-activity phase 2 trial - clinical trial NCT04776629 - is currently being conducted by Abcentra to test the safety and activity of the antibody in patients with severe psoriasis and cardiometabolic risk factors (2). Pre-clinical research of orticumab suggests the possibility of the antibody to inhibit lipoprotein-a (Lp(a)) formation, reduce atherosclerosis, and reduce the thickening of blood vessels.

Orticumab is currently being tested for its ability to reduce atherosclerosis and the thickening of blood vessels. In rodent models, testing has suggested orticumab can reduce the progression of atherosclerosis, regress existing atherosclerotic lesions, and stabilize plaque. In obese rehesus monkeys, orticumab has shown to reduce periperal inflammatory biomarkers (3).

Characterization

BioBricking

We successfully BioBricked our human/mouse anti-oxLDL IgG-encoding device.

Figure 3. Restriction Digest Verification of Anti-oxLDL Plasmid Construct. (A) Virtual digests with BamHI and HindIII on Benchling yield one and two bands at 4.9 kb and 3 kb and 1.9 kb respectively. (B) 1: Anti-oxLDL colony uncut, 1 band below 4.9 kb. 3: Anti-oxLDL cut with HindIII, 3kb, 1.9 kb. 6: Anti-oxLDL cut with BamHI, 1 band at 4.9 kb.

After transformation of the pSB3K3+Labeled anti-oxLDL ligation product into DH5-alpha, plasmids were extracted from 6 white colonies (expected to contain the plasmid of interest) and 1 pink colony (not expected to contain the plasmid of interest) for digestion analysis. In Figure 3, uncut colonies (such as that in lane 1) showed one band lower on the gel than the molecular weight we would expect to see when the plasmid is linearized, indicating the plasmids are high quality and not contaminated with restriction enzymes. Colonies cut with HindIII show two bands at 3 kb and 1.9 kb, as predicted in the virtual digest. Colonies cut with BamHI show one band just under 5 kb, also as predicted.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 826
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1911
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 455