Week 2 HW: Kernel

Part 1: Define your expression target

● Molecule: Human Lactoferrin, image obtained from UniProt (AlphaFold prediction)

● The sequence was obtained from uniprot (P02788 · TRFL_HUMAN), it is a 710 amino acid protein.

Amino Acid sequence:

MKLVFLVLLFLGALGLCLAGRRRSVQWCAVSQPEATKCFQWQRNMRKVRGPPVSCIKRDSPIQCIQAIAENRADAVTLDGGFIYEAGLAPYKLRPVAAEVYGTERQPRTHYYAVAVVKKGGSFQLNELQGLKSCHTGLRRTAGWNVPIGTLRPFLNWTGPPEPIEAAVARFFSASCVPGADKGQFPNLCRLCAGTGENKCAFSSQEPYFSYSGAFKCLRDGAGDVAFIRESTVFEDLSDEAERDEYELLCPDNTRKPVDKFKDCHLARVPSHAVVARSVNGKEDAIWNLLRQAQEKFGKDKSPKFQLFGSPSGQKDLLFKDSAIGFSRVPPRIDSGLYLGSGYFTAIQNLRKSEEEVAARRARVVWCAVGEQELRKCNQWSGLSEGSVTCSSASTTEDCIALVLKGEADAMSLDGGYVYTAGKCGLVPVLAENYKSQQSSDPDPNCVDRPVEGYLAVAVVRRSDTSLTWNSVKGKKSCHTAVDRTAGWNIPMGLLFNQTGSCKFDEYFSQSCAPGSDPRSNLCALCIGDEQGENKCVPNSNERYYGYTGAFRCLAENAGDVAFVKDVTVLQNTDGNNNEAWAKDLKLADFALLCLDGKRKPVTEARSCHLAMAPNHAVVSRMDKVERLKQVLLHQQAKFGRNGSDCPDKFCLFQSETKNLLFNDNTECLARLHGKTTYEKYLGPQYVAGITNLKKCSTSPLLEACEFLRK

● The living system is a yeast (Komagataella phaffii)

● The product needs to be excreted out of the cell to make purification easier.

Part 2: Design your genetic parts

1. I first used the open-source codon optimizer Pichi CLM. I uploaded this DNA sequence into Kernel (Human Lactoferrin codon optimized for Komagataella phaffii). Sequence came from uniprot (P02788 · The full length lactoferin protein seqeunce was used since it requires the full sequence to make an active protein. Komagataella phaffii was chosen as the host because it can reach high cell densities, is widely used for recombinant protein production, and has a eukaryotic secretory pathway capable of producing complex proteins. TRFL_HUMAN) and was codon optimized using open source tool (Pichia CLM). Seqeunce:

ATGAAGCTAGTGTTTTTGGTTTTGCTCTTCTTGGGTGCTTTGGGTTTATGCCTTGCTGGTCGTCGCAGGTCAGTTCAATGGTGTGCTGTCTCCCAACCAGAAGCAACAAAATGTTTTCAGTGGCAAAGAAACATGAGAAAGGTCAGAGGCCCACCAGTTTCATGTATCAAAAGAGACTCTCCAATCCAATGTATTCAAGCTATTGCCGAAAACAGAGCAGACGCCGTGACGCTTGACGGTGGATTTATCTACGAAGCAGGTTTGGCTCCTTACAAACTGAGACCAGTTGCTGCTGAAGTCTACGGTACCGAACGCCAGCCTAGAACTCACTATTACGCTGTCGCTGTCGTCAAGAAGGGTGGTTCTTTCCAGTTGAACGAGTTGCAAGGATTGAAGAGTTGTCATACTGGTTTGCGTCGTACCGCTGGTTGGAACGTGCCCATTGGCACTTTGAGACCATTCCTGAACTGGACTGGTCCACCAGAACCAATCGAAGCCGCAGTGGCCCGTTTCTTCTCTGCTTCATGTGTTCCTGGTGCTGACAAAGGTCAATTCCCTAACTTGTGCCGTTTGTGCGCTGGTACCGGTGAGAACAAATGTGCCTTTTCTTCGCAAGAACCATACTTCAGTTACTCCGGTGCCTTCAAGTGTTTGCGCGATGGAGCTGGAGATGTAGCTTTCATCCGTGAATCCACTGTTTTTGAAGATTTGTCTGATGAAGCTGAAAGAGATGAATACGAGCTGTTATGTCCTGACAATACCCGTAAACCCGTTGACAAGTTCAAGGACTGCCATTTGGCCAGAGTTCCTTCGCATGCTGTTGTAGCTCGTTCCGTCAATGGCAAAGAAGACGCCATTTGGAACTTACTTCGTCAAGCTCAAGAGAAGTTTGGTAAGGATAAGTCTCCTAAGTTCCAGTTGTTTGGTTCTCCATCCGGACAGAAGGATTTGCTGTTCAAAGATTCTGCTATTGGATTTTCTAGAGTCCCACCTAGAATTGATTCTGGTCTTTATTTGGGATCGGGATACTTTACTGCAATTCAAAATTTGAGGAAGAGTGAAGAAGAAGTTGCCGCTCGTCGTGCTAGAGTAGTCTGGTGTGCTGTTGGTGAACAAGAGCTCAGAAAGTGTAACCAATGGAGTGGTCTATCTGAAGGCTCAGTGACTTGCTCTTCTGCATCTACCACTGAAGACTGTATTGCACTGGTACTGAAAGGTGAAGCGGATGCTATGTCTTTGGACGGTGGTTATGTCTATACAGCAGGTAAATGTGGTCTTGTTCCAGTTTTAGCAGAAAATTACAAGAGCCAACAGAGCTCCGACCCTGACCCCAACTGTGTAGATCGTCCCGTTGAAGGCTATCTTGCTGTCGCTGTAGTTAGACGATCCGATACCTCTCTTACATGGAACAGCGTCAAAGGTAAAAAATCTTGTCACACTGCTGTTGACCGTACTGCTGGTTGGAACATCCCTATGGGTTTACTGTTTAACCAGACTGGTTCATGCAAATTTGATGAGTACTTCAGCCAATCTTGTGCTCCAGGATCCGACCCAAGATCCAATTTGTGTGCACTGTGTATCGGTGACGAGCAAGGAGAGAATAAATGTGTCCCAAACTCCAATGAGAGATATTACGGATACACTGGAGCTTTCCGCTGTCTGGCTGAAAATGCCGGAGATGTAGCTTTTGTGAAGGATGTTACTGTTCTCCAAAACACTGACGGTAACAATAATGAAGCTTGGGCTAAGGACCTCAAGTTGGCAGACTTTGCTTTGCTCTGTTTGGATGGGAAGCGTAAACCTGTCACTGAAGCTAGATCGTGCCATCTAGCTATGGCCCCAAACCATGCTGTCGTGTCCAGGATGGACAAAGTTGAGCGTCTCAAGCAAGTGCTTTTGCATCAACAGGCTAAGTTTGGTAGAAATGGATCCGACTGTCCTGACAAGTTCTGTTTGTTCCAATCTGAGACCAAGAATCTATTATTCAATGACAACACTGAATGTCTGGCTAGACTACATGGTAAGACTACCTACGAAAAATACTTGGGTCCCCAATATGTTGCTGGAATCACCAACCTCAAGAAGTGTTCCACTTCTCCACTGTTGGAAGCCTGTGAATTTTTGAGGAAATAA

Part 3: Assemble your construct

I assembled my construct by adding my human lactoferrin sequence immediately downstream of the pPICZ A promoter. Kernel did not let me insert it directly into the pPICZ A backbone, I therefore had to create two separate parts. Part 1: the promoter alone and the pPICZ A backbone without the promoter. There potentially is a better way to do this however, I was not able to find it. Bellow is a schematic of my constructed backbone

Part 4: Optimize and quality-check

I ran the codon optimizer using the lactoferrin AA sequence however it was only for brewers yeast which is a different organism. I got the following sequence back. The sequence change by around 53% (claude analysis), it reduced the GC content.

ATGAAATTGGTTTTTTTGGTTTTGTTGTTTTTGGGTGCTTTGGGTTTGTGTTTGGCTGGTAGAAGAAGATCTGTTCAATGGTGTGCTGTTTCTCAACCAGAAGCTACTAAATGTTTTCAATGGCAAAGAAATATGAGAAAAGTTAGAGGTCCACCAGTTTCTTGTATTAAAAGAGATTCTCCAATTCAATGTATTCAAGCTATTGCTGAAAATAGAGCTGATGCTGTTACTTTGGATGGTGGTTTTATTTATGAAGCTGGTTTGGCTCCATATAAATTGAGACCAGTTGCTGCTGAAGTTTATGGTACTGAAAGACAACCAAGAACTCATTATTATGCTGTTGCTGTTGTTAAAAAAGGTGGTTCTTTTCAATTGAATGAATTGCAAGGTTTGAAATCTTGTCATACTGGTTTGAGAAGAACTGCTGGTTGGAATGTTCCAATTGGTACTTTGAGACCATTTTTGAATTGGACTGGTCCACCAGAACCAATTGAAGCTGCTGTTGCTAGATTTTTTTCTGCTTCTTGTGTTCCAGGTGCTGATAAAGGTCAATTTCCAAATTTGTGTAGATTGTGTGCTGGTACTGGTGAAAATAAATGTGCTTTTTCTTCTCAAGAACCATATTTTTCTTATTCTGGTGCTTTTAAATGTTTGAGAGATGGTGCTGGTGATGTTGCTTTTATTAGAGAATCTACTGTTTTTGAAGATTTGTCTGATGAAGCTGAAAGAGATGAATATGAATTGTTGTGTCCAGATAATACTAGAAAACCAGTTGATAAATTTAAAGATTGTCATTTGGCTAGAGTTCCATCTCATGCTGTTGTTGCTAGATCTGTTAATGGTAAAGAAGATGCTATTTGGAATTTGTTGAGACAAGCTCAAGAAAAATTTGGTAAAGATAAATCTCCAAAATTTCAATTGTTTGGTTCTCCATCTGGTCAAAAAGATTTGTTGTTTAAAGATTCTGCTATTGGTTTTTCTAGAGTTCCACCAAGAATTGATTCTGGTTTGTATTTGGGTTCTGGTTATTTTACTGCTATTCAAAATTTGAGAAAATCTGAAGAAGAAGTTGCTGCTAGAAGAGCTAGAGTTGTTTGGTGTGCTGTTGGTGAACAAGAATTGAGAAAATGTAATCAATGGTCTGGTTTGTCTGAAGGTTCTGTTACTTGTTCTTCTGCTTCTACTACTGAAGATTGTATTGCTTTGGTTTTGAAAGGTGAAGCTGATGCTATGTCTTTGGATGGTGGTTATGTTTATACTGCTGGTAAATGTGGTTTGGTTCCAGTTTTGGCTGAAAATTATAAATCTCAACAATCTTCTGATCCAGATCCAAATTGTGTTGATAGACCAGTTGAAGGTTATTTGGCTGTTGCTGTTGTTAGAAGATCTGATACTTCTTTGACTTGGAATTCTGTTAAAGGTAAAAAATCTTGTCATACTGCTGTTGATAGAACTGCTGGTTGGAATATTCCAATGGGTTTGTTGTTTAATCAAACTGGTTCTTGTAAATTTGATGAATATTTTTCTCAATCTTGTGCTCCAGGTTCTGATCCAAGATCTAATTTGTGTGCTTTGTGTATTGGTGATGAACAAGGTGAAAATAAATGTGTTCCAAATTCTAATGAAAGATATTATGGTTATACTGGTGCTTTTAGATGTTTGGCTGAAAATGCTGGTGATGTTGCTTTTGTTAAAGATGTTACTGTTTTGCAAAATACTGATGGTAATAATAATGAAGCTTGGGCTAAAGATTTGAAATTGGCTGATTTTGCTTTGTTGTGTTTGGATGGTAAAAGAAAACCAGTTACTGAAGCTAGATCTTGTCATTTGGCTATGGCTCCAAATCATGCTGTTGTTTCTAGAATGGATAAAGTTGAAAGATTGAAACAAGTTTTGTTGCATCAACAAGCTAAATTTGGTAGAAATGGTTCTGATTGTCCAGATAAATTTTGTTTGTTTCAATCTGAAACTAAAAATTTGTTGTTTAATGATAATACTGAATGTTTGGCTAGATTGCATGGTAAAACTACTTATGAAAAATATTTGGGTCCACAATATGTTGCTGGTATTACTAATTTGAAAAAATGTTCTACTTCTCCATTGTTGGAAGCTTGTGAATTTTTGAGAAAATGATAA

Prior to pasting my sequence into kernel, I had already used an open source codon optimizer for my organism (Pichi CLM). Therefore I did not change my sequence in kernel since the I had used a more fitting tool.

Biosecurity Risk Using Kernels tool I did not find any biosecurity risks.

Blast By blasting my sequence the only thing that came up was Sitophilus oryzae transferrin (LOC115881357), mRNA.

Part 5: From design to build

I would order the CDS as one synthetic gene (2,136 bp), buy the vector, and join them with Gibson. I would purchase the pPICZ A backbone. I would join the two using Gibson assembly. Gibson assembly is preferred since the complexity is low and does not necessitate a more complex approach.

Sequence difficulty isn’t my constraint: no repeats of 20 bp or more, GC 37 to 55% across every 100 nt window, longest homopolymer six A’s, and the plasmid’s two bad AT runs are in the promoter and terminator I’m buying.

Once I construct my plasmid using gibson I would transfect it into E Coli to produce it. I would then extract it and sequence it to make sure that it was inserted well into the bacbone. I would then transfect it into yeast and try producing my product.

Part 6: What don’t you know yet?

Whether my signal peptide works. I kept the native human secretion peptide, but I couldn’t find one paper expressing human lactoferrin in Pichia with it they all used α-factor. I’d build five versions differing only in the first 57 bp and measure secreted protein by ELISA.

Whether it folds. Codon optimization does nothing for disulfides, and mature lactoferrin has 32 cysteines in 16 of them plus two iron lobes. A band at the right size proves nothing, so I’d check iron binding by A465/A280.

Readings: Carr et al. 2004, NAR 32:e162 · Gibson et al. 2009, Nat Methods 6:343 · Kosuri et al. 2010, Nat Biotechnol 28:1295