Week 2: DNA Read Write and Edit
Part 0: Basics of Gel Electrophoresis I watch the lecture on live and the recorded recitation video, However, when I wanted to review rhw recorded video ofr the lecture the link did not exist on the HTGAA 2026 page. However, we got via email a recording of the 2025 lecture.
Part 1: Benchling & In-silico Gel Art Intructions: See this week’s lab protocol “Gel Art: Restriction Digests and Gel Electrophoresis” for details. Overview:
Make a free account at benchling.com [Maxmatusbenchling](https://benchling.com/organizations/maxmatus)
Import the Lambda DNA (https://www.neb.com/en/-/media/nebus/page-images/tools-and-resources/interactive-tools/dna-sequences-and-maps/text-documents/lambdagbk.txt?rev=50c75f4579114750a9ad75d892d7d118&hash=EF15DDE468761F64D50E30418917B08D).
Simulate Restriction Enzyme Digestion with the following Enzymes:(https://benchling.com/xquenda_lab/enzyme-lists/22892)
EcoRI
HindIII
BamHI
KpnI
EcoRV
SacI
SalI
Create a pattern/image in the style of Paul Vanouse’s Latent Figure Protocol artworks.
You might find Ronan’s website a helpful tool for quickly iterating on designs!
**Part 3: DNA Design Challenge**
I choosed the protein P04439 · HLAA_HUMAN from the UniProt Database (https://www.uniprot.org/uniprotkb?query=Human)
I decide to choose this protein becouse it relates with human inmmunity and my finnal project is about how to develop a biomaterial to protect humans from some bacterias, so I found interesting this protein as starting point. Some of the characteristics assosiated to this protein according to UniProt are:
HLA class I histocompatibility antigen, A alpha chain · Gene: HLA-A (HLAA) · Homo sapiens (Human) · 365 amino acids · Evidence at protein level
The sequence of the P04439 · HLAA_HUMAN protein is:
>sp|O95905|ECD_HUMAN Protein ecdysoneless homolog OS=Homo sapiens OX=9606 GN=ECD PE=1 SV=1
MEETMKLATMEDTVEYCLFLIPDESRDSDKHKEILQKYIERIITRFAPMLVPYIWQNQPF NLKYKPGKGGVPAHMFGVTKFGDNIEDEWFIVYVIKQITKEFPELVARIEDNDGEFLLIE AADFLPKWLDPENSTNRVFFCHGELCIIPAPRKSGAESWLPTTPPTIPQALNIITAHSEK ILASESIRAAVNRRIRGYPEKIQASLHRAHCFLPAGIVAVLKQRPRLVAAAVQAFYLRDP IDLRACRVFKTFLPETRIMTSVTFTKCLYAQLVQQRFVPDRRSGYRLPPPSDPQYRAHEL GMKLAHGFEILCSKCSPHFSDCKKSLVTASPLWASFLESLKKNDYFKGLIEGSAQYRERL EMAENYFQLSVDWPESSLAMSPGEEILTLLQTIPFDIEDLKKEAANLPPEDDDQWLDLSP DQLDQLLQEAVGKKESESVSKEEKEQNYDLTEVSESMKAFISKVSTHKGAELPREPSEAP ITFDADSFLNYFDKILGPRPNESDSDDLDDEDFECLDSDDDLDFETHEPGEEASLKGTLD NLKSYMAQMDQELAHTCISKSFTTRNQVEPVSQTTDNNSDEEDSGTGESVMAPVDVDLNL VSNILESYSSQAGLAGPASNLLQSMGVQLPDNTDHRPTSKPTKN
Reverse Translate results
Results for 644 residue sequence “sp|O95905|ECD_HUMAN Protein ecdysoneless homolog OS=Homo sapiens OX=9606 GN=ECD PE=1 SV=1” starting “MEETMKLATM”
Reverse translation of sp|O95905|ECD_HUMAN Protein ecdysoneless homolog OS=Homo sapiens OX=9606 GN=ECD PE=1 SV=1 to a 1932 base sequence of most likely codons. atggaagaaaccatgaaactggcgaccatggaagataccgtggaatattgcctgtttctg attccggatgaaagccgcgatagcgataaacataaagaaattctgcagaaatatattgaa cgcattattacccgctttgcgccgatgctggtgccgtatatttggcagaaccagccgttt aacctgaaatataaaccgggcaaaggcggcgtgccggcgcatatgtttggcgtgaccaaa tttggcgataacattgaagatgaatggtttattgtgtatgtgattaaacagattaccaaa gaatttccggaactggtggcgcgcattgaagataacgatggcgaatttctgctgattgaa gcggcggattttctgccgaaatggctggatccggaaaacagcaccaaccgcgtgtttttt tgccatggcgaactgtgcattattccggcgccgcgcaaaagcggcgcggaaagctggctg ccgaccaccccgccgaccattccgcaggcgctgaacattattaccgcgcatagcgaaaaa attctggcgagcgaaagcattcgcgcggcggtgaaccgccgcattcgcggctatccggaa aaaattcaggcgagcctgcatcgcgcgcattgctttctgccggcgggcattgtggcggtg ctgaaacagcgcccgcgcctggtggcggcggcggtgcaggcgttttatctgcgcgatccg attgatctgcgcgcgtgccgcgtgtttaaaacctttctgccggaaacccgcattatgacc agcgtgacctttaccaaatgcctgtatgcgcagctggtgcagcagcgctttgtgccggat cgccgcagcggctatcgcctgccgccgccgagcgatccgcagtatcgcgcgcatgaactg ggcatgaaactggcgcatggctttgaaattctgtgcagcaaatgcagcccgcattttagc gattgcaaaaaaagcctggtgaccgcgagcccgctgtgggcgagctttctggaaagcctg aaaaaaaacgattattttaaaggcctgattgaaggcagcgcgcagtatcgcgaacgcctg gaaatggcggaaaactattttcagctgagcgtggattggccggaaagcagcctggcgatg agcccgggcgaagaaattctgaccctgctgcagaccattccgtttgatattgaagatctg aaaaaagaagcggcgaacctgccgccggaagatgatgatcagtggctggatctgagcccg gatcagctggatcagctgctgcaggaagcggtgggcaaaaaagaaagcgaaagcgtgagc aaagaagaaaaagaacagaactatgatctgaccgaagtgagcgaaagcatgaaagcgttt attagcaaagtgagcacccataaaggcgcggaactgccgcgcgaaccgagcgaagcgccg attacctttgatgcggatagctttctgaactattttgataaaattctgggcccgcgcccg aacgaaagcgatagcgatgatctggatgatgaagattttgaatgcctggatagcgatgat gatctggattttgaaacccatgaaccgggcgaagaagcgagcctgaaaggcaccctggat aacctgaaaagctatatggcgcagatggatcaggaactggcgcatacctgcattagcaaa agctttaccacccgcaaccaggtggaaccggtgagccagaccaccgataacaacagcgat gaagaagatagcggcaccggcgaaagcgtgatggcgccggtggatgtggatctgaacctg gtgagcaacattctggaaagctatagcagccaggcgggcctggcgggcccggcgagcaac ctgctgcagagcatgggcgtgcagctgccggataacaccgatcatcgcccgaccagcaaa ccgaccaaaaac
Reverse translation of sp|O95905|ECD_HUMAN Protein ecdysoneless homolog OS=Homo sapiens OX=9606 GN=ECD PE=1 SV=1 to a 1932 base sequence of consensus codons. atggargaracnatgaarytngcnacnatggargayacngtngartaytgyytnttyytn athccngaygarwsnmgngaywsngayaarcayaargarathytncaraartayathgar mgnathathacnmgnttygcnccnatgytngtnccntayathtggcaraaycarccntty aayytnaartayaarccnggnaarggnggngtnccngcncayatgttyggngtnacnaar ttyggngayaayathgargaygartggttyathgtntaygtnathaarcarathacnaar garttyccngarytngtngcnmgnathgargayaaygayggngarttyytnytnathgar gcngcngayttyytnccnaartggytngayccngaraaywsnacnaaymgngtnttytty tgycayggngarytntgyathathccngcnccnmgnaarwsnggngcngarwsntggytn ccnacnacnccnccnacnathccncargcnytnaayathathacngcncaywsngaraar athytngcnwsngarwsnathmgngcngcngtnaaymgnmgnathmgnggntayccngar aarathcargcnwsnytncaymgngcncaytgyttyytnccngcnggnathgtngcngtn ytnaarcarmgnccnmgnytngtngcngcngcngtncargcnttytayytnmgngayccn athgayytnmgngcntgymgngtnttyaaracnttyytnccngaracnmgnathatgacn wsngtnacnttyacnaartgyytntaygcncarytngtncarcarmgnttygtnccngay mgnmgnwsnggntaymgnytnccnccnccnwsngayccncartaymgngcncaygarytn ggnatgaarytngcncayggnttygarathytntgywsnaartgywsnccncayttywsn gaytgyaaraarwsnytngtnacngcnwsnccnytntgggcnwsnttyytngarwsnytn aaraaraaygaytayttyaarggnytnathgarggnwsngcncartaymgngarmgnytn garatggcngaraaytayttycarytnwsngtngaytggccngarwsnwsnytngcnatg wsnccnggngargarathytnacnytnytncaracnathccnttygayathgargayytn aaraargargcngcnaayytnccnccngargaygaygaycartggytngayytnwsnccn gaycarytngaycarytnytncargargcngtnggnaaraargarwsngarwsngtnwsn aargargaraargarcaraaytaygayytnacngargtnwsngarwsnatgaargcntty athwsnaargtnwsnacncayaarggngcngarytnccnmgngarccnwsngargcnccn athacnttygaygcngaywsnttyytnaaytayttygayaarathytnggnccnmgnccn aaygarwsngaywsngaygayytngaygaygargayttygartgyytngaywsngaygay gayytngayttygaracncaygarccnggngargargcnwsnytnaarggnacnytngay aayytnaarwsntayatggcncaratggaycargarytngcncayacntgyathwsnaar wsnttyacnacnmgnaaycargtngarccngtnwsncaracnacngayaayaaywsngay gargargaywsnggnacnggngarwsngtnatggcnccngtngaygtngayytnaayytn gtnwsnaayathytngarwsntaywsnwsncargcnggnytngcnggnccngcnwsnaay ytnytncarwsnatgggngtncarytnccngayaayacngaycaymgnccnacnwsnaar ccnacnaaraay
3.4. You have a sequence! Now what?
What technologies could be used to produce this protein from your DNA? Describe in your words the DNA sequence can be transcribed and translated into your protein. You may describe either cell-dependent or cell-free methods, or both.
To answere this question I used Copilot and privided the sequence that I got from the reverse translation of sp|O95905|ECD_HUMAN Protein. The reply was as follow:
Cell‑dependent systems:
Bacterial expression (e.g., E. coli) using a plasmid vector with a strong promoter (like T7).
Yeast or insect cells for eukaryotic expression.
Mammalian cells (e.g., HEK293, CHO) if proper human‑like folding or modifications are important.
Cell‑free systems:
E. coli lysate–based systems,
Wheat germ or rabbit reticulocyte lysates,
Or a PURE system with purified transcription/translation components.
In all cases, the same basic logic applies: the DNA encodes the amino‑acid sequence of ECD via triplet codons.
In my understanding the most easy and adfordable way to produce this protein would be a cell‑dependent systems in E. coli using a plasmid vector with a strong promoter. However, I still have to make a critic analyzis of this Copilot answere according to the most uptated literature.
Part 4: Prepare a Twist DNA Synthesis Order
4.1. Create a Twist account and a Benchling account
Maxmatusbenchling I created a Twist account with my personal mail, however, when I am trying to login a window pop-up asking me to contact my local privider in Mexico. Nevertheless, it looks like the webpage of the local provider is not working, I tryed to charg it many times unsuscesfully, so I was not able to finish this secction of the homework: [Twist_Mexico] (https://ecommerce.twistdna.com/www.abalat.com.mx)
4.2. Build Your DNA Insert Sequence
4.3. On Twist, Select The “Genes” Option
4.4. Select “Clonal Genes” option
4.5. Import your sequence
4.6. Choose Your Vector
Part 5: DNA Read/Write/Edit
5.1 DNA Read
(i) What DNA would you want to sequence (e.g., read) and why?
I would like to sequence the grape pomace DNA in order to understan if there are some antibacterial proteins in it.
(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:
Is your method first-, second- or third-generation or other? How so?
What is your input? How do you prepare your input (e.g. fragmentation, adapter ligation, PCR)? List the essential steps.
What are the essential steps of your chosen sequencing technology, how does it decode the bases of your DNA sample (base calling)?
What is the output of your chosen sequencing technology?
5.2 DNA Write
(i) What DNA would you want to synthesize (e.g., write) and why?
If I find antibacterial proteins in grape pomenace I would like to mass produce it in order to enhance this property and then insert it into a biocuir made out of grape pomenace. I found this web page wich seems to be very usefull for my project: Grape_genome and also an interesting article related to the use of pomenace as antimicrobial in feed: Antimicrobial_pomenace
(ii) What technology or technologies would you use to perform this DNA synthesis and why? Also answer the following questions:
What are the essential steps of your chosen sequencing methods?
What are the limitations of your sequencing method (if any) in terms of speed, accuracy, scalability?
5.3 DNA Edit
(i) What DNA would you want to edit and why?
Not sure yet
(ii) What technology or technologies would you use to perform these DNA edits and why? Also answer the following questions:
How does your technology of choice edit DNA? What are the essential steps?
What preparation do you need to do (e.g. design steps) and what is the input (e.g. DNA template, enzymes, plasmids, primers, guides, cells) for the editing?
What are the limitations of your editing methods (if any) in terms of efficiency or precision?