Imre's SynBio recources for advanced cloners

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table of contents


Background

I work mainly with E. coli as a chassis for gene regulatory networks. Gene regulatory networks are programs based on logic (information wise) and DNA/RNA/protein interactions (as executables). The program is generally encoded on DNA (a plasmid) and read by the host organism for execution. Thus,the methods presented here are not comprehensive and serve the specific purpose of constructing synthetic DNA and working with single celled organisms and their products. Furthermore, I will mainly give keywords and short teasers of each method for you to pick and follow up on.

Software

Google Scholar

The search engine for scientific literature. Here you can find almost all publications. Many however are behind a paywall. [1]

Sci-Hub

Every scientist should dedicate their thesis to Alexandra Elbakyan. Sci hub is a link to servers in Kazakhstan, which, by the deeds of ingenious hacker/knowledge liberator Alexandra Elbakyan, host most of the paywalled papers you will find on google scholar. As some organizations regard Sci-Hub as a criminal website, it changes domain frequently - however, it remains easy to find using common search engines.

Benchling

An online tool to organize, design, and register synthetice DNA (plasmids, primers etc) and organisms. It allows you to annotate/copy/paste sequences and finds restriction enzyme cutting sites and more. It also allows you to keep a journal and upload/link to files and standard protocols. There is a free and commercial option. I use it as a base for all my planning before ordering any DNA or starting an experiment. [2]

UniProt

UniProt is a comprehensive repositora for protein sequences, structure, genomic and functional data. Good to find gene sequences of a protein you heard about and want to play with. [3]

EcoCyc

A dedicated website to E. coli, the to date best understood and most used organism in research. It has sequences, metabolic networks, strains and more. [4]

FPbase - the fluorescent protein database

A beautyful website, listing fluorescent reporter proteins, with spectra, sequence and gnealogy. [5]

NCBI - National Center for Biotechnology Information

The central database for sequences, organism genomes, functions, structures etc. It has a nice search function, where for instance you can paste a DNA sequence and search for the translated proteine sequence (to find homologues in different organisms) and vice versa. [6]

Expasy - the Swiss Institute of Bioinformatics

Similar to NCBI it is a huge database, but swiss and with a very nice web design. It has some great protein models, if you are into that. [7]

Wetware

PCR - polymerase chain reaction

After 2021, you know what this is. You may not know, that you can use it to add new sequences to a given template by including them in your primer. A primer which anneals to a target but has additional bases (the overhang) will add those to the final PCR product. You can stack PCR reactions about three times, adding additional basses to the piece like that before unspecific byproduct will make your amplicon unusable. These overhangs are also used for Gibson assembly or homologous recombination. If your PCR primers differ greatly in melting temperatures, you can also do a two step PCR, first 5 cycles with low melting temperature and then the rest with the higher. This will create a cleaner product with less byproducts.

Gibson assembly

The now famous Daniel Gibson came up with the idea of stitching plasmids and PCR parts together by sequence specific overhangs. Each overhang is a barcode, ensuring each piece goes to the right place. The practice is very neat but kinda costly.

Homologous recombination

If you plan on working with yeat, you can use this method to insert your synthetic gene into the yeat genome, making it more stable without the need for antibiotic seleciton. It is done by including flanking regions, to your gene insert, homologous to a sequence within the yeast genome. The native DNA repair then inserts the gene at this site. There is a bit more to that, so read up on the specifics.

MEGAWHOP - megaprimer whole plasmid amplification

If Gibson assembly is too costly and you have either only a few inserts or the time to do sequential cloning. MEGAWHOP uses the overhangs of a PCR amplicon, homologous to a plasmid sequence, to amplify the entire plasmid, using the PCR amplicon as a large primer. This creates a new plasmid with two nicks, which are effortlessly repaired by the host organism after transformation.

CRISPRi - CRISPR inhibition

A deactivated Cas9 (dCas9) binds to a target sequence but doesnt cut it. If the target is a promoter, it functionally silences or decreases the gene expression from this promoter. If you create a host strain constantly expressing dCas9, you only need to transform a new guide RNA (gRNA) to target a new gene. As it is not knocking out the gene entirely, it can also be used for essential gene knock down / temporary knock out by making the gRNA transcription inducible.

Autolysate cell free transcription and translation

Cell free transcription and translation (TxTl) is a quick and versatile method to prototype and test genetic parts, without the entire cloning and transformation steps. It is basically an in vitro expression system which can read and express genes from a PCR amplicon, giving a fast feedback on a possible part before constructing the whole network. However, it is costly and difficult to manufacture TxTl extracts. The autolysate E. coli strain has an integrated functionality which makes it much easier to create TxTl extracts, without the use of a sonicator, "only" using a (ultra)centrifuge.