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• Most antibody databases are woefully incomplete so they actually aren't a great place to look for antibodies.  They  However, they do give you a good idea of what kind of antigens people are raising antibodies against, though.
• If you have an antigen in mind, searching PDB can get you to some antibody names.  Then use those antibody names to do a deeper literature search to see if you can find the sequence or binding site information you need.
• Cross-check peptide sequences from companies that sell your antigens to make sure the peptide sequence matches that of the antibody's binding site.  Try to do this early on or you might be sad.

My rationale for recommending the alternative antibodies HyHEL-10 (anti-hen egg lysozyme) and VRC01 (anti-HIV1 gp120):

• Chicken lysozyme and HIV1 gp120 as antigens:
  • There are many antibodies against both of these antigens, a .  A number of which those are monoclonal, sequenced, and have been studied by NMR or crystallography, so there are .  This gave me multiple options to choose from.
  • They are commercially available for not too expensive (chicken lysozyme is a cheap enzyme and gp120 is about the price of an antibody).
  • They can probably be handled at BSL1 or at least at BSL2 (gp120 can probably be handled at BSL1 since active HIV can be handled at BSL2 and gp120 alone shouldn't infect cellsbe infectious).
  • They should have minimal effects on HEK293 cells (hen egg lysozyme works on cleaves bacterial cell walls and gp120 binds CD4, which is an immune receptor so probably not expressed at high levels in HEK293).
  • Chicken lysozyme has been studied extensively in appears to be an exsiting model system for investigating antigen-antibody interactions (so it's a good model system) and detecting HIV1 gp120 could be useful in gene/cell therapy against HIV.
• HyHEL-10 as an antibody:
  • Sequence available
  • Binding site information available (and corresponds to the sequence of commercially available hen egg white lysozyme)
  • Potential problem:  antibody originally generated in mouse
• VRC01 as an antibody:
  • Sequence available
  • Broadly neutralizing - According to the discovery study, this antibody neutralized the sera of about 90% of HIV patients tested, which means that even though commercially available HIV gp120 peptides come with a variety of sequences this antibody will probably bind them all.
  • Binding kinetics are available for various strains of HIV1 (and numbers are good for modeling!)
  • Some binding site information is available (alanine scanning)
  • Potential problem:  some  there are some small discrepancies between sequences of commercially available gp120 peptides and the proteins used in the studyliterature (see below for details)

I still have 0.5beta on this page as a backup antibody for gp120.  VRC01  However, VRC01 is better though suited to our application because it can recognize gp120 from many different strains.  Also,  The other problem is that the epitope recognized by 0.5beta has a recognition sequence that isn't in exactly in commercially-available peptides.  There's a one amino acid substitution which hasn't been studied in the literature.one amino acid different from commercially available gp120 peptides.  There is no literature on 0.5beta binding to this modified epitope as far as I know.

CLONING STRATEGY:

gBlocks and cloning steps are in Geneious:

grapefruit > iGEM2014 > Sandboxes > BCR 2.0 > New antibodies > (antibody name) > (heavy/light chain)

Amino Cloning steps for each heavy and light chain are in Geneious in the "New antibodies folder" under "BCR 2.0".  Amino acid sequences were back-translated using IDT, optimizing for gBlocks 's gBlock optimization algorithm (there were no BsaI cut sites to eliminateremove).Since we

The heavy chain gBlocks that are in those folders only contain the variable region.  I think we should PCR the constant region from our Gmab heavy plasmid for a number of reasons:

1. We had issues with mutations when we ordered the gantenerumab heavy chain as one gBlock

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1. , probably because of the length of the gBlock.
2. Ordering the entire heavy chain costs about $240 more (per sample) than just ordering the heavy chain variable region

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1. .
2. We'll need a way of PCRing out the

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1. constant region

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1. if we eventually want to make an expression vector that has the constant region already built in anyway (to facilitate variable region swapping).  That said, since we're only testing a couple of antibodies

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1. for now, I think

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1. making

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1. a vector

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1. like this should wait until later

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1. .

Primers for the mIgM the linkers where we are testing so many of them).  Primers for that PCR are in the "IgM heavy chain primers" folder.  The primer regions primers don't make it look like we'll have too many problems...VRC01 and HyHEL-10 gBlocks are located in their respective folders, along with the pENTR that they will make when ligated into the Golden Gate backbonethey should cause us too much trouble...

BUDGET:

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