The rationale used in designing parts is as follows

Acinetobacter baylyi ADP1 is an interesting organism for its natural competency and its ability to degrade aromatic compounds and lignocellulosic mass. Factors influencing Expression of protein:(Glick et al 1987)^[1]

• The strength of the promoter
• The efficiency of RBS
• Substate cofactor availability
• The half life of mRNA
• The metabolic state of the cell
• Stability of the foreign protein in the cell
• The abundance of the specific tRNA/Codon Usage table
• The copy number of the gene encoding the protein of interest
• 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, ie, Promoters, RBS and codon usage table.
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 [6].

For our project, we used T5 promoter[BBa_K592008]. Being a viral promoter, it is constitutive and is known to express in a wide range of hosts including E. coli*(Hermann Bujard, Reiner Gentz, Michael Lanzer, Dietrich Stueber, Michael Mueller, Ibrahim Ibrahimi, Marie-Therese aeuptle, Bernhard Dobberstein, [26] A T5 promoter-based transcription-translation system for the analysis of proteins in Vitro and in Vivo, Methods in Enzymology, Academic Press, Volume 155, 1987, Pages 416-433)* and A. baylyi. Another advantage is that the promoter library that we create would 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 A. baylyi ADP1.

We have classified the promoters we made 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. We kept the nucleotides immediately next to -35 and -10 regions also same so as to ease the binding of RNA polymerase to the promoter. Care was also taken not to accidentally insert a biobrick restriction or Afl(II) restriction site. 4 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 from Mordaka P. M. et al 2018.

6 promoters were created using this approach: (BBa_K2857007, BBa_K2857008, BBa_K2857009, BBa_K2857010, BBa_K2857011, 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 promtors, we used Salis lab RBS calculator to calculate RBS specific for each promoter, GFP and Acinetobacter baylyi ADP1.
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. There was one table available on kasuza 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 ***(check)***1174 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 do not replicate in A. baylyi ADP1. So, in the literature, we found pBAV1k which works in A. baylyi. We PCR amplified the backbone such that it amplifies the vector without GFP. So that we can clone GFP in that place. For promoter studies, we amplified it such that region from BioBrick prefix to GFP part is amplified but the promoter region is not so that we can clone synthesized promoter there for our studies.
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 has high copy number and it replicated in a wide range of host (all gram-negative bacteria and some Gram-positive ones).
GFP was cloned in pBAV1k downstream of T5 promoter and RBS. Then fluorometry experiments were carried out. Similarly, Promoters were cloned upstream GFP in pBAV1k and then fluorometry studies were carried out.

1. Lorem ipsum dolor sit amet
2. Lorem ipsum dolor sit amet
3. Lorem ipsum dolor sit amet
4. Lorem ipsum dolor sit amet
5. Lorem ipsum dolor sit amet
6. Lorem ipsum dolor sit amet
7. Lorem ipsum dolor sit amet
8. Lorem ipsum dolor sit amet
9. Lorem ipsum dolor sit amet, consectetur adipiscing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Interdum posuere lorem ipsum dolor. Morbi tristique senectus et netus et. .