Team:IIT-Madras/Results

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Team: IIT-Madras/Results

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Results

Promoters result

From 20 promoters, we got successful cloning results for 15 promoters.
We did the fluorometry results for these 15 promoters.
The promoter names are P1R, P3R, P4R, P5R, P6R, P70R, P8R, P9R, P71R, P1S, P2S, P5S, P6S, P8S, P71S.
We took pBAV1k-T5-GFP(BBa_K1321309) as control which we got from addgene(https://www.addgene.org/26702/). On the other hand, wild-type is taken as negative control.
All promoters were cloned from pBAV1k vector backbone under GFP. Then fluorometry studies were carried out with each clone. From these 15 promoters, we got a spectrum of promoters showing varying expressions. R-Series promoters (Promoters having Salis lab calculated RBS) mostly exhibit better expression rates than S series.

For each promoter, we took two biological replicate and three technical replicates. The errors and differences in each of them are reflected in the error bars.

Promoters showing more expression than T5 Promoters P8R(BBa_K2857116), P9R(BBa_K2857119), P1R(BBa_K2857111), P1S(BBa_K2857101), and P3R(BBa_K2857113) showed higher expression than the T5 promoter.

Promoters P5R(BBa_K2857115), P5S(BBa_K2857105), P2S(BBa_K2857102), P71R(BBa_K2857120), P4R(BBa_K2857114), P6R(BBa_K2857116), P70R(BBa_K2857117),P71S(BBa_K2857) and P6S(BBa_K2857106) show lower expression than the T5 promoter.

P18 and T5 promoter P18 shows extremely high expression of GFP about more than 75 fold higher
Conclusion:

  • We have been successful in getting a spectrum of promoters which is desirable for a synthetic promoter library generation.
    • We got promoters having higher expression than T5 promoter as well as ones having lower expression than T5.
  • We also have promoter P18 which gives about 75 fold higher expression than T5 promoter
P18 and T5 promoter P18 shows extremely high expression of GFP about more than 75 fold higher.

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Codon Usage Table obtained from CUTE for Acinetobacter baylyi ADP1 and Fluorescent protein

From the Acinetobacter baylyi ADP1 genome, we took 1194 genes which we obtained after removing hypothetical and putative sequences from the coding sequences annotation available from NCBI. The Codon Usage Table Enumerator (CUTE) tool from ChassiDex is used to generate the codon-usage table for the organism.
The occurrence of each codon is shown below(Fig1) in the genome of A. baylyi
The table attached below lists the percentage distribution of different codons among the analyzed genes.
The table attached below gives the probability of a particular amino acid being coded for by a particular codon.
These tables were then used to codon-optimize the eGFP and mCherry reporter proteins using sequences available from NCBI. Codon-optimized GFP was restriction cloned into the pBAV1K vector and transformed into A. baylyi. It showed promising results as illustrated below, with the codon-optimized reporter showing considerably higher fluorescence than the control. The magnitudes of the fluorescence readings on the y-axis are depicted in arbitrary units relative to pBAV1k-T5-GFP(BBa_K1321309) in Acinetobacter baylyi ADP1. WT is wild-type strain of Acinetobacter baylyi ADP1 while the Last one shows Fluorometry data for Acinetobacter baylyi ADP1 with codon optimized GFP in pBAV1k under the T5 promoter. The error bars are calculated by taking into account two biological replicates and three technical replicates of each biological replicate. Conclusion:

  • From the fluorometry, we can clearly see that Codon optimized GFP shows higher expression than other GFP. This clearly demonstrates that Expression of codon optimized GFP is higher than GFP.
  • This suggests that Codon optimization has been successful.
  • This also suggests that the Codon usage table generated using CUTE is successful.