The truth is not always what it appears to be

This is a myth. With our gene synthesis protocol we can not only synthesise genes of any length, we can also synthesise complete plasmids.

Synthetic genes are often created by assembling overlapping DNA oligonucleotides. With this method it is possible to create long fragments that will be finally assembled to one large synthetic gene. By adding a circularisation procedure it is therefore possible to even create complete plasmids of any length. As we routinely work with E. coli, your “gene” (i.e. the new plasmid) must harbour an E.coli selection marker and an origin of replication, of course.

Still the question remains, why someone would like to create his / her own plasmid. There are quite a few answers to that, like:

• Free choice in antibiotic resistance, promoters, origin of replication, etc.

• Free choice of the multiple cloning site, tailor-made for your projects

• No potential licensing issues if you want to use your plasmid commercially

• Sequence optimization of the vector to the host organism, to regulate the expression levels of the gene(s) (e.g. antibiotic resistance etc.)

The fact is:

With our proprietary SDM technology we can efficiently mutate, insert or delete up to 150 bp at the target site within your plasmid. If you only want to target one site, e.g. to test a different amino acid in the active domain of your protein, Standard SDM is the method of choice. And if you have more than just one target site, SDM also is the perfect way to get the multiple-sites-mutated plasmids very fast and cost efficient. With our multi SDM technology we can simultaneously mutate up to 5 sites in just one round. And, of course, also these multiple mutations can be up to 6 bp mutations, insertions or deletions

Due to the redundant nature of the DNA code more than one triplet codon can code for an amino acid e.g. Arginine - CGT, CGC, CGA, CGG, AGA, AGG. The frequency of these codons differs across species. It is therefore possible to optimize a DNA sequence so it contains the same frequency of codons as seen in the organism where the gene will be expressed. The optimization also helps in the reduction of high GC regions and repeat regions in the gene.

This type of optimization is only possible however when the gene to be synthesized (or part of it) is coding for a protein sequence and therefore has codons that can be manipulated in this way. A non-coding stretch of DNA does not contain codons coding for an amino acid and therefore an optimization exploiting the redundancy of DNA cannot be done.

The fact is:

Eurofins’ optimisation software GENEius does not only use the best codons. We have shown that this very simple approach does not result in highest protein expression.

During the optimisation process GENEius randomly as­sembles the DNA sequence and then analyses it in relation to codon usage by comparing it to the input codon usage table. This input codon usage table is usually taken from the Kazusa Codon Usage Database (http://www.kazusa.or.jp/codon), but it can also be provided by the customer. GENEius does not simply aim for a high codon adaption index (CAI), instead it harmonises the codons used. Frequently used codons from highly expressed genes are more often used in the resulting gene sequence than less frequently used codons. Very rare codons, however, will be completely avoided. During adap­tion GENEius also checks for “bad motifs” like restriction sites and avoids artificial splice sites, unspecific transcription factor binding sites, etc. Also, to minimise RNA structure direct and in­verted repeats are avoided as they not only make synthesis more difficult, they can decrease DNA stability and reduce efficiency of transcription and translation in E.coli. And last but not least, the GC content is equally distributed to avoid GC-rich subse­quences within in the gene. All these parameters are taken into account and an “optimisation score” is constantly being calculated. If this score falls below a certain threshold, the sequence is taken as the output. This procedure results in a different DNA sequence every time the optimisation is running.

This is a fact, indeed. You only need to cut your vector of choice with one or two suitable enzymes of the MCS. Your gene insert can then be PCR amplified with primers that generate homology to the vector at the ends of the PCR product. These homologous regions can then be used for subcloning via SLIC (= sequence and ligation independent cloning) into your vector. Any gene internal restriction sites do not matter at all because the PCR product won’t be cut during subcloning. Eurofins’ molecular biology experts will do the design and make sure that all your requirements are met, e.g., in frame cloning. SLIC is as fast and efficient as traditional subcloning via restriction enzyme sites and, if you have forgotten, e.g., to introduce a stop codon or a fusion tag at the end of your gene, this can also be fixed simultaneously.

The fact is:

Well, yes, most scientists  use gene synthesis for expression improvement of their genes of choice in heterologous systems. This can be achieved via codon usage adaptation using Eurofins’ gene adaption and optimisation software “GENEius”. But there are many more advantages for using gene synthesis. Imagine your NGS data reveal a very interesting DNA sequence that you now want to analyse further. With gene synthesis you can simply order your DNA or RNA sequence, you just need to know the in silico sequence. Another advantage of gene synthesis is fast and reliable access to cDNA sequences that a few years ago would have been done via labour-, time- and cost-intensive cDNA synthesis. No RNA extraction, no RT-PCR, and no RACE is needed for getting perfect full-length cDNA when using gene synthesis. Unwanted restriction sites can be avoided during adaption and subcloning into your own expression vector is also possible. Of course you can combine your gene sequence together with promoter and enhancer elements, polyadenylation signals, restriction sites, etc.

Well, yes, this is a fact (but please see our tip below!). If you only order your open reading frame with restriction sites, this is true. Our pEX vectors are based on pUC18 and do not contain a promoter or terminator for expression of your subcloned gene. They are standard cloning vectors for E.coli. For transcription and translation experiments the gene would have to be subcloned into an expression vector of your choice, harbouring a suitable promoter and potentially a terminator sequence. By the way, for best expression results the ORF should be optimised using our powerful algorithm “GENEius”.

And here is a tip for saving time and money:

Include a promoter sequence upstream of your gene sequence. Of course you could also add a terminator sequence at the 3’ end. Then, the pEX vector can be used as an expression vector for protein expression in vivo or, after linearisation, as template for in vitro transcription.

The fact is:

The standard vectors from Eurofins have been designed to have few restriction enzymes in the multiple cloning site (MCS). Even if your restriction enzyme is present in the MCS this will not cause a problem. The extra fragment will be so small that it will not be visible on an agarose gel. All restriction sites in the MCS are at least 4bp (in most cases >10bp) away from your gene so the efficiency of the restriction digest will also not be affected.

In the very unlikely event that one of your chosen restriction sites is also in the vector backbone and a band the same or similar length as your gene is present after the digest, there are a number of possibilities to get around this problem.

Either a 3^rd enzyme can be used in the digest that cuts in the vector backbone reducing the size of the unwanted band. In the majority of cases the required band can then be easily gel eluted from an agarose gel.

If this strategy does not work due to the lack of an appropriate restriction site then the gene can be amplified with PCR primers. It is then possible to digest the PCR product with the required enzymes and proceed with the subcloning. It is important in this case to add 2-3 bases to the 5´ ends of the PCR primers as overhangs so the efficiency of the digest is not reduced.