The rationale:

In synthetic biology and metabolic engineering studies the level of protein expression is very important and may be phase dependent, inducible or constitutive. There are various factors influencing proteins expression levels:(Glick et al 1987) ^[1]

• The strength of the promoter^[2]
• The efficiency of RBS^[3]
• Substate cofactor availability^[4]
• The half life of mRNA^[5]
• The metabolic state of the cell
• Stability of the foreign protein in the cell
• The abundance of the specific tRNA/Codon Usage table^[6]
• The copy number of the gene encoding the protein of interest^[7]
• Protein interaction with other proteins in a chassis
• Presence of inducer or signalling molecule for Inducible promoter

In our project, we focused on three factors - Promoters, RBS and a codon usage table.

Promoter: It is the region upstream of the gene where RNA polymerase binds and initiates transcription. The binding of RNA polymerase to the promoter is often the rate-limiting step in a bacterial system, as translation and transcription are coupled, unlike an eukaryotic system where the mRNA undergoes post-transcriptional modifications. Hence the promoter sequence is the major translational regulator of gene expression.

RBS: It is the region just upstream of the coding region that binds to the Ribosomal unit and initiates translation. The mRNA conformation at RBS is extremely detrimental in bringing both subunits of ribosome together and initiate translation.

Codon usage: Each species have a different abundance of tRNA. Hence, each species have a different preference for each codon for each amino acids. The abundance of tRNA levels can also make difference in translational rates. By process of Codon optimization, expression of genes can be increased by multiple folds.


Promoters (using random and rational approaches): Promoters usually have a construct as shown below.

In region 1, it has typically about 17 Nucleotides, then the -35 conserved region which is recognized by RNA polymerase while transcription initiation. Then follows Region 2 which is about 17 nucleotides long. -10 follows which is typically TATA box. Another motif that is conserved for promoters. Then follows 6 nucleotides long Region which we named region 3 and then there is a transcription initiation site. This usually starts with A. From this site Transcription starts.

The regions flanking -35 and -10 regions ie Region 1,2 and 3 affect the strength of the promoter ^[8].

For our project, we used the T5 promoter [BBa_K592008]. T5 is a constitutive promoter that is not under the influence of any protein. It is known to work in a broad range of microorganisms including E. coli, Acinetobacter baylyi ADP1etc. ^[9]. Another advantage of creating T5 promoter-based library is that they might also work in other chassis like E. coli, other industrially important chassis like Cornybacterium glutamicum. However, documentation and characterization of these promoters in other chassis would be required. We have characterized these promoters in Acinetobacter baylyi ADP1.

We have classified the promoters we build based on their designing methods into two categories:
i)P category: Randomizing the nucleotides flaking -35 and -10 region by conserving their GC content percentage wise in individual regions. Nucleotides flanking -35 and -10 regions were also kept same so as to keep the interaction of RNA polymerase with binding sites unaltered. Care was also taken not to accidentally insert a biobrick restriction or Afl(II) restriction site.
We designed Four promoters from this category were made (BBa_K2857003, BBa_K2857004, BBa_K2857005, BBa_K2857006)
ii)Q Category: In this approach, we introduced point mutations in BBa_K592008 promoter sequence (in silico). These promoters were selected based on the percentage of similarity they have with T5 BBa_K592008 promoter. Care was taken not to create any restriction site or introduce mutation on -35 and -10 regions. Based on that we selected the promoters having 57% similarity, 66%, 79%, 85%, and 91%. We selected two 79% similarity promoters with different sequences. This Method was inspired by Mordaka P. M. et al 2018 ^[10].

We designed Six promoters were created using this approach (Q5:BBa_K2857007, Q6:BBa_K2857008, Q70:BBa_K2857009, Q71:BBa_K2857010, Q9:BBa_K2857011, Q8:BBa_K2857012)
Since, this library is created out of T5 promoter, which is known to work in multiple chassis, the library that we created should also work in various chassis like almost all E. coli strains, A. baylyi, L. lactis.

For each of the 10 promoters, we used Salis lab RBS calculator to calculate RBS specific for each promoter, GFP and Acinetobacter baylyi ADP1.(https://salislab.net/software/) (Salis, H M. “The Ribosome Binding Site Calculator.” Methods in Enzymology., U.S. National Library of Medicine, www.ncbi.nlm.nih.gov/pubmed/21601672.). These RBS have been named as Biobrick BBa_K2857013-BBa_K2857022.
Next, we assembled the RBS with promoter sequences and got the complete DNA sequence synthesized (BioBrick BBa_K2857111-BBa_K2857120).
The same promoters under iGEM RBS are (BBa_K2857101-BBa_K2857110), which also we got synthesized from the IDT and submitted.

When we contacted GenScript for sending us codon optimized GFP, they did not have reliable data on codon usage table of Acinetobacter baylyi ADP1. There was one table available on kasuza(https://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=202950) which is based only on two CDS.
We identified this industry based problem that the codon optimizers that they have codon usage table data mainly for standard hosts which are widely used. This is a hindrance in using the unconventional host for one’s studies. So, we created CUTE on the chassidex website. It can be found on CUTE ChassiDex. This online free tool can be used to generate Codon usage of any organism as long as its CDS annotation is available.
We used CUTE to generate Codon usage table data for A. baylyi ADP1 by taking into consideration the CDS annotation available on the NCBI site. This table can be found on the Results page of our wiki. This table is generated by taking into account at least 1194 CDS. We removed putative and hypothetical proteins from the data used to generate the table.
Using this codon usage table, we Codon optimized eGFP and mCherry for Acinetobacter baylyi ADP1. We have submitted these Biobrick(BBa_K2857001 GFP and BBa_K2857002 mCherry).

iGEM standard vectors pSB1C3 does not replicate in A. baylyi ADP1. From the literature, we found that pBAV1k works in A. baylyi^[11]. We amplified pBAV1k reporters less version. For promoter studies, we amplified pBAV1k so as to get promoterless vector backbone where our promoters can be cloned. Similarly, for reporter studies, we amplified pBAV1k such that, we could get reporter-less vector backbone where codon optimized GFP can be placed.
pBAV1k could not be submitted due to the material-transfer agreement but it can be purchased from ADD GENE(https://www.addgene.org/26702/). This is also added as BioBrick (BBa_K1321309). This vector is a high copy, broad host range vector.
GFP was cloned in pBAV1k downstream of the T5 promoter and RBS. To measure the strength of Promoter we conducted fluorometry experiments were carried.
Similarly, Promoters were cloned upstream GFP in pBAV1k. To measure the codon-optimized GFP then fluorometry studies were carried out to find out the expression levels.

References