Week 2 homework

DNA read, write, and edit 🧬

Part 1: Benchling and in-silico gel art

The genome of the λ-phage was imported and virtually digested with the following restriction endonucleases: EcoRI, HindIII, BamHI, KpnI, EcoRV, SacI, and SalI before being visualized on Benchling’s agarose gel simulator (Figure 2.1).

Figure 2.1 Virtual digest of λ-phage’s DNA treated with seven different restriction enzymes (as indicated by the gel lane legend on the top left). Figure created on Benchling.com.

Part 3: DNA design challenge

3.1. Choose your protein. In recitation, we discussed that you will pick a protein for your homework that you find interesting. Which protein have you chosen and why? Using one of the tools described in recitation (NCBI, UniProt, Google), obtain the protein sequence for the protein you chose.

The entire medical establishment relies heavily on a sea creature so ancient and resilient that it has barely changed since its ancestral form first appeared in the tree of life when Planet Earth had its own ring system (like Saturn and Uranus), approximately 450 million years ago. Besides a hardy armour-like exoskeleton and a bizarre body plan, horseshoe crabs bring to the table a primitive, yet extremely functional, immune system tightly-packed in their blue-colored blood. A key component of their immune response are the granular amoebocytes found in their blood, which, upon contact with a bacterial endotoxin, initiate a coagulation cascade that protects the horseshoe crab by sequestering and neutralizing the harmful agent. This very property of horseshoe crab immunity has been harnessed by numerous medical corporations since the 1960s-1970s as an effective method to safely screen vaccines, other injectable pharmaceuticals, as well as implantable biomedical devices, for the presence of bacteria-derived toxins ¹.

Based on this premise, an interesting idea to pursue would be to perform whole-cell engineering of bacteria or, preferably, yeast cells to render them functionally similar to the granular amoebocytes contained in horseshoe crab blood. This endeavor, primarily inspired by an old iGEM project aiming to convert Escherichia coli bacteria into red blood cells ², could contribute to the conservation of the fragile ecosystem to which horseshoe crabs belong, but also drastically confine the invasive, time-consuming, and expensive practice of harvesting horseshoe crab haemolymph ³.

To this end, a critical step is to transform the cells-to-be-engineered with the gene coding for factor C, namely the main protein that initiates the immune response and triggers the coagulation pathway ^{4 5}. The amino acid sequence for the factor C protein of the mangrove horseshoe crab Carcinoscorpius rotundicauda (CrFactor C) was retrieved from UniProt under the accession number “Q26422” ^{4 6}:

>sp|Q26422|LFC_CARRO Limulus clotting factor C OS=Carcinoscorpius rotundicauda OX=6848 PE=2 SV=1 MVLASFLVSGLVLGLLAQKMRPVQSKGVDLGLCDETRFECKCGDPGYVFNIPVKQCTYFY RWRPYCKPCDDLEAKDICPKYKRCQECKAGLDSCVTCPPNKYGTWCSGECQCKNGGICDQ RTGACACRDRYEGVHCEILKGCPLLPSDSQVQEVRNPPDNPQTIDYSCSPGFKLKGMARI SCLPNGQWSNFPPKCIRECAMVSSPEHGKVNALSGDMIEGATLRFSCDSPYYLIGQETLT CQGNGQWNGQIPQCKNLVFCPDLDPVNHAEHKVKIGVEQKYGQFPQGTEVTYTCSGNYFL MGFDTLKCNPDGSWSGSQPSCVKVADREVDCDSKAVDFLDDVGEPVRIHCPAGCSLTAGT VWGTAIYHELSSVCRAAIHAGKLPNSGGAVHVVNNGPYSDFLGSDLNGIKSEELKSLARS FRFDYVRSSTAGKSGCPDGWFEVDENCVYVTSKQRAWERAQGVCTNMAARLAVLDKDVIP NSLTETLRGKGLTTTWIGLHRLDAEKPFIWELMDRSNVVLNDNLTFWASGEPGNETNCVY MDIQDQLQSVWKTKSCFQPSSFACMMDLSDRNKAKCDDPGSLENGHATLHGQSIDGFYAG SSIRYSCEVLHYLSGTETVTCTTNGTWSAPKPRCIKVITCQNPPVPSYGSVEIKPPSRTN SISRVGSPFLRLPRLPLPLARAAKPPPKPRSSQPSTVDLASKVKLPEGHYRVGSRAIYTC ESRYYELLGSQGRRCDSNGNWSGRPASCIPVCGRSDSPRSPFIWNGNSTEIGQWPWQAGI SRWLADHNMWFLQCGGSLLNEKWIVTAAHCVTYSATAEIIDPNQFKMYLGKYYRDDSRDD DYVQVREALEIHVNPNYDPGNLNFDIALIQLKTPVTLTTRVQPICLPTDITTREHLKEGT LAVVTGWGLNENNTYSETIQQAVLPVVAASTCEEGYKEADLPLTVTENMFCAGYKKGRYD ACSGDSGGPLVFADDSRTERRWVLEGIVSWGSPSGCGKANQYGGFTKVNVFLSWIRQFI

3.2. Reverse Translate: Protein (amino acid) sequence to DNA (nucleotide) sequence. The Central Dogma discussed in class and recitation describes the process in which DNA sequence becomes transcribed and translated into protein. The Central Dogma gives us the framework to work backwards from a given protein sequence and infer the DNA sequence that the protein is derived from. Using one of the tools discussed in class, NCBI or online tools (Google “reverse translation tools”), determine the nucleotide sequence that corresponds to the protein sequence you chose above.

The nucleotide coding sequence for CrFactor C was extracted from the respective NCBI entry ⁷:

>S77063.1 factor C=endotoxin-sensitive intracellular serine protease zymogen {clone CrFC21} [Carcinoscorpius rotundicauda=Singapore horseshoe crabs, blood, amoebocytes, CDS, 3060 nt] ATGGTCTTAGCGTCGTTTTTGGTGTCTGGTTTAGTTCTAGGGCTACTAGCCCAAAAAATGCGCCCAGTTCAGTCCAAAGGAGTAGATCTAGGCTTGTGTGATGAAACGAGGTTCGAGTGTAAGTGTGGCGATCCAGGCTATGTGTTCAACATTCCAGTGAAACAATGTACATACTTTTATCGATGGAGGCCGTATTGTAAACCATGTGATGACCTGGAGGCTAAGGATATTTGTCCAAAGTACAAACGATGTCAAGAGTGTAAGGCTGGTCTTGATAGTTGTGTTACTTGTCCACCTAACAAATATGGTACTTGGTGTAGCGGTGAATGTCAGTGTAAGAATGGAGGTATCTGTGACCAGAGGACAGGAGCTTGTGCATGTCGTGACAGATATGAAGGGGTGCACTGTGAAATTCTCAAAGGTTGTCCTCTTCTTCCATCGGATTCTCAGGTTCAGGAAGTCAGAAATCCACCAGATAATCCCCAAACTATTGACTACAGCTGTTCACCAGGGTTCAAGCTTAAGGGTATGGCACGAATTAGCTGTCTCCCAAATGGACAGTGGAGTAACTTTCCACCCAAATGTATTCGAGAATGTGCCATGGTTTCATCTCCAGAACATGGGAAAGTGAATGCTCTTAGTGGTGATATGATAGAAGGGGCTACTTTACGGTTCTCATGTGATAGTCCCTACTACTTGATTGGTCAAGAAACATTAACCTGTCAGGGTAATGGTCAGTGGAATGGACAGATACCACAATGTAAGAACTTGGTCTTCTGTCCTGACCTGGATCCTGTAAACCATGCTGAACACAAGGTTAAAATTGGTGTGGAACAAAAATATGGTCAGTTTCCTCAAGGCACTGAAGTGACCTATACGTGTTCGGGTAACTACTTCTTGATGGGTTTTGACACCTTAAAATGTAACCCTGATGGGTCTTGGTCAGGATCACAGCCATCCTGTGTTAAAGTGGCAGACAGAGAGGTCGACTGTGACAGTAAAGCTGTAGACTTCTTGGATGATGTTGGTGAACCTGTCAGGATCCACTGTCCTGCTGGCTGTTCTTTGACAGCTGGTACTGTGTGGGGTACAGCCATATACCATGAACTTTCCTCAGTGTGTCGTGCAGCCATCCATGCTGGCAAGCTTCCAAACTCTGGAGGAGCGGTGCATGTTGTGAACAATGGCCCCTACTCGGACTTTCTGGGTAGTGACCTGAATGGGATAAAATCGGAAGAGTTGAAGTCTCTTGCCCGGAGTTTCCGATTCGATTATGTCCGTTCCTCCACAGCAGGTAAATCAGGATGTCCTGATGGATGGTTTGAGGTAGACGAGAACTGTGTGTACGTTACATCAAAACAGAGAGCCTGGGAAAGAGCTCAAGGTGTGTGTACCAATATGGCTGCTCGTCTTGCTGTGCTGGACAAAGATGTAATTCCAAATTCGTTGACTGAGACTCTACGAGGGAAAGGGTTAACAACCACGTGGATAGGATTGCACAGACTAGATGCTGAGAAGCCCTTTATTTGGGAGTTAATGGATCGTAGTAATGTGGTTCTGAATGATAACCTAACATTCTGGGCCTCTGGCGAACCTGGAAATGAAACTAACTGTGTATATATGGACATCCAAGATCAGTTGCAGTCTGTGTGGAAAACCAAGTCATGTTTTCAGCCCTCAAGTTTTGCTTGCATGATGGATCTGTCAGACAGAAATAAAGCCAAATGCGATGATCCTGGATCACTGGAAAATGGACACGCCACACTTCATGGACAAAGTATTGATGGGTTCTATGCTGGTTCTTCTATAAGGTACAGCTGTGAGGTTCTCCACTACCTCAGTGGAACTGAAACCGTAACTTGTACAACAAATGGCACATGGAGTGCTCCTAAACCTCGATGTATCAAAGTCATCACCTGCCAAAACCCCCCTGTACCATCATATGGTTCTGTGGAAATCAAACCCCCAAGTCGGACAAACTCGATAAGTCGTGTTGGGTCACCTTTCTTGAGGTTGCCACGGTTACCCCTCCCATTAGCTAGAGCAGCCAAACCTCCTCCAAAACCTAGATCCTCACAACCCTCTACTGTGGACTTGGCTTCTAAAGTTAAACTACCTGAAGGTCATTACCGGGTAGGGTCTCGAGCCATCTACACGTGCGAGTCGAGATACTACGAACTACTTGGATCTCAAGGCAGAAGATGTGACTCTAATGGAAACTGGAGTGGTCGGCCAGCGAGCTGTATTCCAGTTTGTGGACGGTCAGACTCTCCTCGTTCTCCTTTTATCTGGAATGGGAATTCTACAGAAATAGGTCAGTGGCCGTGGCAGGCAGGAATCTCTAGATGGCTTGCAGACCACAATATGTGGTTTCTCCAGTGTGGAGGATCTCTATTGAATGAGAAATGGATCGTCACTGCTGCCCACTGTGTCACCTACTCTGCTACTGCTGAGATTATTGACCCCAATCAGTTTAAAATGTATCTGGGCAAGTACTACCGTGATGACAGTAGAGACGATGACTATGTACAAGTAAGAGAGGCTCTTGAGATCCACGTGAATCCTAACTACGACCCCGGCAATCTCAACTTTGACATAGCCCTAATTCAACTGAAAACTCCTGTTACTTTGACAACACGAGTCCAACCAATCTGTCTGCCTACTGACATCACAACAAGAGAACACTTGAAGGAGGGAACATTAGCAGTGGTGACAGGTTGGGGTTTGAATGAAAACAACACCTATTCAGAGACGATTCAACAAGCTGTGCTACCTGTTGTTGCAGCCAGCACCTGTGAAGAGGGGTACAAGGAAGCAGACTTACCACTGACAGTAACAGAGAACATGTTCTGTGCAGGTTACAAGAAGGGACGTTATGATGCCTGCAGTGGGGACAGTGGAGGACCTTTAGTGTTTGCTGATGATTCCCGTACCGAAAGGCGGTGGGTCTTGGAAGGGATTGTCAGCTGGGGCAGTCCCAGTGGATGTGGCAAGGCGAACCAGTACGGGGGCTTCACTAAAGTTAACGTTTTCCTGTCATGGATTAGGCAGTTCATTTGA

3.3. Codon optimization. Once a nucleotide sequence of your protein is determined, you need to codon optimize your sequence. You may, once again, utilize Google for a “codon optimization tool”. In your own words, describe why you need to optimize codon usage. Which organism have you chosen to optimize the codon sequence for and why?

Different organisms display different codon biases regarding protein translation ^{8 9}. Codon bias and codon usage are predominantly determined by the relative abundance of aminoacyl-tRNAs and aminoacyl-tRNA synthetases inside a cell, as they both constitute crucial components of the translational machinery, carrying the proteinogenic amino acids and loading the aminoacyl-tRNAs with the proper amino acid respectively. This codon bias has to be taken into account when transforming an organism with a gene from another organism to modify the coding sequence accordingly, enabled by the degeneracy of the genetic code, and render it compatible with the host cell’s translational machinery, thus ensuring smooth heterologous expression of the protein of interest. If the gene of interest is not codon optimized for the expression host, it is likely that the protein will be synthesized at very low levels or not at all.

Similarly, when choosing an expression host for synthesizing the protein of interest, several parameters have to be considered as well. In this specific case, factor C is a protein derived from a eukaryote, has a complex molecular structure involving disulfide bonds, and is glycosylated at several amino acid positions. For the expression of a protein with those characteristics, a putative host should also be a eukaryote, as eukaryotic cells harbor the necessary biochemical pathways for protein post-translational modifications, such as glycosylation, and, additionally, should have an oxidizing intracellular environment to facilitate the formation of disulfide bridges. A promising candidate that fulfils all those criteria is the methylotrophic yeast Pichia pastoris, for which the original coding sequence for factor C has been codon optimized employing Benchling’s codon optimization tool:

>factor C=endotoxin-sensitive intracellular serine protease zymogen {clone CrFC21} [codon optimized for Pichia pastoris, CDS, 3060 nt] ATGGTCTTAGCGTCGTTTTTGGTTTCTGGTTTAGTTCTAGGGCTACTAGCCCAAAAAATGCGCCCAGTTCAGTCCAAAGGAGTAGATCTAGGCTTGTGTGATGAAACGAGGTTCGAGTGTAAGTGTGGCGATCCAGGCTATGTTTTCAACATTCCAGTCAAACAATGCACATACTTTTATCGATGGAGGCCGTATTGTAAACCATGTGATGACCTGGAGGCTAAGGATATTTGTCCAAAGTACAAGCGATGTCAAGAGTGTAAGGCTGGTCTTGATAGTTGTGTTACTTGTCCACCTAACAAGTATGGTACTTGGTGTAGCGGTGAATGTCAGTGCAAGAACGGAGGTATCTGTGACCAGAGGACAGGAGCTTGTGCATGTCGTGACAGATATGAAGGGGTGCACTGCGAAATTCTCAAAGGTTGTCCTCTTCTTCCATCGGATTCTCAGGTTCAAGAAGTCAGAAATCCACCAGATAATCCCCAAACTATTGACTACAGCTGCTCACCAGGGTTCAAGCTTAAGGGTATGGCACGAATTAGCTGCCTCCCAAATGGACAGTGGAGTAACTTTCCACCAAAATGTATTAGAGAATGTGCCATGGTTTCATCTCCAGAACATGGTAAAGTTAATGCTCTTTCCGGTGATATGATAGAAGGTGCTACTTTACGGTTCTCCTGTGATAGTCCCTACTACTTGATTGGTCAAGAAACATTAACCTGCCAAGGTAATGGTCAGTGGAATGGACAGATACCACAATGTAAGAACTTGGTCTTTTGCCCTGACCTGGATCCTGTAAACCATGCTGAACACAAGGTTAAAATTGGTGTTGAACAAAAATATGGTCAGTTTCCTCAAGGAACTGAAGTTACCTATACGTGTTCGGGTAACTACTTCTTGATGGGTTTTGATACCTTAAAATGCAACCCTGATGGGTCTTGGTCAGGATCACAGCCATCCTGTGTTAAAGTGGCAGACAGAGAGGTCGACTGTGACAGTAAAGCTGTAGACTTCTTGGATGATGTTGGTGAACCGGTCAGGATCCACTGTCCTGCTGGCTGTTCTTTGACAGCTGGTACTGTTTGGGGTACAGCCATATACCATGAGCTTTCCTCCGTGTGCCGCGCAGCCATCCATGCTGGCAAGCTTCCAAACTCTGGAGGAGCTGTCCATGTTGTGAACAATGGCCCGTACTCCGACTTTCTGGGTTCCGACCTGAATGGTATAAAATCGGAAGAGTTGAAGTCTCTTGCCAGAAGTTTTAGATTCGATTATGTCCGTTCCTCCACAGCAGGTAAGTCAGGATGCCCTGATGGATGGTTTGAGGTAGACGAGAACTGTGTGTATGTTACATCAAAGCAGAGAGCATGGGAAAGAGCTCAAGGTGTGTGCACCAATATGGCTGCTAGACTTGCTGTGCTGGACAAAGATGTAATTCCAAACTCGTTGACTGAGACTCTAAGAGGGAAAGGTTTAACCACCACGTGGATAGGATTGCATAGACTAGATGCTGAGAAGCCCTTTATTTGGGAGTTAATGGATCGTAGTAATGTGGTTCTGAATGATAACCTAACCTTCTGGGCCTCTGGTGAACCTGGAAATGAAACTAACTGCGTATATATGGACATCCAAGATCAGTTGCAGTCTGTGTGGAAAACCAAGTCATGTTTTCAGCCATCTAGTTTTGCTTGCATGATGGATCTGTCAGATAGAAATAAAGCCAAGTGCGATGATCCTGGATCATTGGAAAATGGACACGCCACACTTCATGGACAATCCATTGATGGTTTCTATGCTGGTTCTTCTATAAGGTACAGCTGCGAGGTTCTCCACTACCTCAGTGGAACTGAAACCGTAACTTGTACCACAAATGGCACTTGGAGTGCTCCGAAACCGCGATGTATCAAAGTCATCACCTGCCAAAACCCCCCTGTACCATCATATGGTTCTGTGGAAATCAAACCCCCAAGTAGAACTAACTCGATAAGTCGTGTTGGGTCACCTTTCTTGAGGTTGCCAAGATTACCCCTCCCATTAGCTAGAGCAGCCAAGCCTCCTCCAAAGCCTAGATCCTCACAACCCTCTACTGTGGACTTGGCCTCTAAGGTTAAATTGCCTGAAGGTCATTACCGTGTCGGGTCTAGGGCCATCTACACGTGCGAGTCGAGATACTACGAACTATTGGGATCTCAAGGCAGAAGATGTGACTCTAACGGAAACTGGTCCGGTCGGCCAGCGAGCTGTATTCCAGTTTGCGGACGGTCAGATTCTCCTCGTTCTCCTTTTATCTGGAATGGTAATTCTACAGAAATTGGTCAGTGGCCGTGGCAGGCAGGAATCTCTAGATGGCTTGCAGACCACAATATGTGGTTTCTCCAATGTGGAGGATCTCTATTGAATGAGAAGTGGATCGTCACTGCTGCCCATTGTGTCACCTACTCTGCTACTGCTGAGATTATTGACCCCAATCAATTTAAAATGTATCTGGGCAAGTACTACCGTGATGACTCCAGAGATGATGACTATGTACAAGTAAGAGAGGCTCTTGAGATCCACGTCAATCCTAACTACGACCCCGGCAATTTGAACTTTGACATAGCCTTGATTCAACTGAAAACTCCTGTTACTTTGACTACACGAGTCCAACCAATTTGTCTGCCTACTGACATCACGACAAGAGAACATTTGAAGGAGGGAACATTAGCAGTTGTTACGGGTTGGGGTTTGAATGAAAACAACACCTATTCAGAGACTATTCAACAAGCTGTGTTGCCTGTTGTTGCAGCCAGCACCTGCGAAGAGGGGTACAAGGAGGCAGACTTACCACTGACTGTTACAGAGAACATGTTCTGTGCAGGTTACAAGAAGGGACGTTATGATGCCTGCTCCGGTGACAGCGGAGGACCTTTAGTGTTTGCTGATGATTCCCGTACCGAAAGGAGATGGGTCTTGGAAGGGATTGTCAGCTGGGGCAGTCCCTCCGGATGTGGAAAGGCGAACCAGTATGGTGGCTTCACTAAAGTTAACGTTTTCCTGTCATGGATTAGACAATTCATTTAA

3.4. You have a sequence! Now what? What technologies could be used to produce this protein from your DNA? Describe in your words how the DNA sequence can be transcribed and translated into your protein. You may describe either cell-dependent or cell-free methods, or both.

For the expression of the codon-optimized version of CrFactor C in P. pastoris, the first step would be to replace the regulatory elements integrated in the cassette generated above, as they have been selected for bacterial expression, with parts that would be recognized in a yeast cell, such as the methanol-activated AOX1 promoter, an appropriate Kozak sequence, and the AOX1 terminator. After assembling the new genetic cassette for P. pastoris expression, it would have to be inserted into an integrative vector (probably from the pPICZα series), which would also carry a selection marker, for instance, zeocin, in conjunction with the expression cassette for the protein of interest. Subsequently, this integrative vector would be employed for the transformation of the yeast cells. In a portion of the successfully transformed yeast cells, the expression cassette-selection marker sequence would then be incorporated into the organism’s genome and, through the antibiotic resistance conferred by the selection marker, those positive transformants could be identified. By utilizing zeocin in particular, the most highly expressing strains can be readily isolated through increasing the dose of the antibiotic, as the resistance provided by zeocin is directly proportional to the number of selection marker genes integrated, which is a direct indication for the number of genes encoding the protein of interest integrated as well. The highly-expressing positive transformants would, afterwards, be cultured in the presence of methanol, which can strongly induce the transcription of the codon-optimized CrFactor C gene, whose mRNA would then be translated (ensured by the Kozak consensus) into CrFactor C protein molecules. Lastly, the nascent CrFactor C protein would be transferred to the endoplasmic reticulum (ER) and the Golgi apparatus for post-translational modifications.

For proteins with extended post-translational modification requirements, such as factor C, a cell-free expression system would not be recommended. However, a T7-based in vitro transcription method, coupled with a highly active and reliable in vitro eukaryotic translation system, such as rabbit reticulocyte lysate (RRL), would be an appealing alternative. For the expression of recombinant CrFactor C, though, the translation system should also be supplemented with microsomal membranes to secure the capacity for glycosylation.

Part 4: Prepare a Twist DNA Synthesis Order

By following the instructions on preparing a DNA synthesis order for Twist, a plasmid containing the CrFactor C sequence codon-optimized for P. pastoris was generated (Figure 2.2).

Part 5: DNA read/write/edit

5.1 DNA Read (i) What DNA would you want to sequence (e.g., read) and why? This could be DNA related to human health (e.g. genes related to disease research), environmental monitoring (e.g., sewage waste water, biodiversity analysis), and beyond (e.g. DNA data storage, biobank).

In continuation of the CrFactor C project, once again, expressing the codon-optimized sequence in P. pastoris would require assembling the genetic cassette, including the gene and all its flanking regulatory elements. Sequencing the assembled construct constitutes an important step before proceeding with the transformation in order to verify that the cloning was indeed successful and that the newly assembled expression cassette is identical to the designed one.

(ii) In lecture, a variety of sequencing technologies were mentioned. What technology or technologies would you use to perform sequencing on your DNA and why?

Also answer the following questions:

1. Is your method first-, second- or third-generation or other? How so?
2. What is your input? How do you prepare your input (e.g. fragmentation, adapter ligation, PCR)? List the essential steps.
3. What are the essential steps of your chosen sequencing technology, how does it decode the bases of your DNA sample (base calling)?
4. What is the output of your chosen sequencing technology?

For whole-plasmid sequencing of the integrative vector carrying the CrFactor C codon-optimized sequence, I would choose Oxford Nanopore sequencing. It is a third-generation sequencing technology that combines high speed, reliability, and read accuracy (98-99%), as well as low cost.