Team:IIT-Madras/Notebook

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Team: IIT-Madras/Results

Notebook

13 April: We attempted making competant E. coli DH5alpha cells. We got the strain from Guhan sir's lab
16 April: We tried checking its competancy by RFP transformation but not so good results.
20 April: We again tried making Competant cells but we did not liquid nitrogen for flash freeze step. we use SOC media instead of LB this time.
22 April: We tried transformation but it fails in some plates. Much fewer colonies.
26 April:We meet Guhan sir and Anantha barathi ma'am for guidance and help. Also, try to understand feasibility of the project.

8 May: Exams get over and we discusses ideas with Anantha ma'am(Instructor). One interesting study could be lignocellulosic mass conversion to other useful and economically high valued products.
8-10 May: We do and extensive literature survey for this project and Acinetobacter baylyi ADP1 seems to be good candidates.
14 May: We finalise to work on lignin conversion to biofuels using Acinetobacter baylyi ADP1. We choose this organisms over other because, it is non pathogenic and has amazing capabilities of degrading aromatic compounds like caffeate, vaillate, etc. Above all, it is naturally competent and Guhan sir's lab have expertise in working with this organism.
15 May: We finalize to work on lignin conversion to biofuels using Acinetobacter baylyi ADP1. We choose this organisms over other because, it is non pathogenic and has amazing capabilities of degrading aromatic compounds like caffeate, vaillate, etc. Above all, it is naturally competant and Guhan sir's lab have expertise in working with this organism.
16 May: Acinetobacter baylyi has 6 copies of transposonal elemets which were remove in 2017 and a new strain of this organism was engineered called ADP-!Sx (Suárez GA, Renda BA, Dasgupta A, Barrick JE. Reduced Mutation Rate and Increased Transformability of Transposon-Free Acinetobacter baylyi ADP1-ISx. Kivisaar M, ed. Applied and Environmental Microbiology. 2017;83(17):e01025-17. doi:10.1128/AEM.01025-17.) We request barrick to send us the engineered strain.
18 May: We plan to compile all literature and decide for direction of the project. Vanillin production seems to be potential candidate but experiments fail.
22 May: We realise that we need a proper toolkit and well established methods to work with A. baylyi. So we plan our project to take it into direction of Expanding the toolbox of A. baylyi ADP1.
23 May: We finalise upon the things we want to try out: Codon optimized Reporters and promoter. If time permits then terminator. 25 May: Anantha ma'am asks sagar to place order of CO GFP and mCherry for now to GenScript Meanwhile we do a lit survey for various approahes for promoter design.
29 May: GenScript gets back to us with a reply that there is no reliable codon usage table available. They send us link of kasuza website which has a codon table based on 2 coding regions.
30 May: We knew that coding region annotation was present on NCBI website. we discussed this problem with KD and idea of CUTE was born.
31 May: Workplan was made to start working on Promoter Design, Codon usage table on A. baylyi and Codon optimization. We fixed upon using pBAV1K vector as it is known to work in A. baylyi and sagar had preliminary results for it KD, burhan sathvik were assigned on Codon table of A. baylyi
Richa, Burhan, Sathvik get on Promoter design.

4 June: Codon generator was ready to be used on chassidex website. We took the complete CDS annotation of A. baylyi and removed putative and hypothetical regions. We were left with 1174 CDS regions. We got the codon usage information. The data was then normalized and Codon table of A. baylyi was generated.
5 June: We now had enough Data to have some parameters for designing promoters. Richa helped us to find regions upstream of the housekeeping genes in order to locate the -35 and -10 regions.
6 June: We tried talking to Anindhya Sir of Medha bioinformatics but even then, we could not find conserved regions. Meanwhile, we codon optimize the GFP and mcherry using the codon table. We removed the less used codons with frequently used codons and screened for unwanted restriction sites. Then we send the sequence to Anantha ma'am for one more eye check.
8 June: We use various other tools such as clustalW, MEME suite, etc but could not find the consensus regions upstream of the genes that we selected with not much success.
10 June: We discuss about generic nature of CUTE and plan to implement same in the promoter sequences. We also discussed the nature of pBAV1K vector that can replicate in so many host organism.
11 June: We discuss and agree upon making a T5 promoter based library for our project.
12 June: GFP and mCherry designs were corrected where-ever necessary. Planned to place order for it.
13 June: Literature survey of T5 promoter and promoter based library started.
18 June: Lab training period for Burhan and sathvik started. Anantha barathi ma'am, Sagar, Velvizhi Devi, Suchi smita helped out in Lab training period. Various basic molecular biology and microbiology techniques were taught.This training made us familiar with lab Safety, lab rules, familiaring with lab equipments was done during this period In parallel, we exploring various methods and approaches for building T5 library.
19 June: We placed order for Codon optimized GFP and mCherry to IDT. We finalized upon the parameters and start generating sequences.
22-26 June: Sathvik leave for home, lab training continues. Burhan generates promoter library containing over 300 sequences of same design. Meanwhile since, project abstract deadline was approaching, we decided to give a nice name. Sathvik named our project 'ADaPtat1on- Expanding the toolkit of Acinetobacter baylyi.
29 June: Burhan leaves and lab training gets over

9 July: We discuss over the phone about Clostridium Synthetic promoter library paper that was published recently.Seeing we also try to incorporate that approach in some of our promoters out of complete promoter library.
10 July Parameters taken into consideration by authors were taken into consideration and similar lines were followed to generate such library 16 July We have the promoter sequences from both categories. Former being called P and later as Q. We plan to add Salis lab calculated RBS also to the promoters
16 July Codon optimized reporters arrives.
18 July We assembled the parts in silico and then screened for unwanted restriction sites and agreed down on selection of part of each catergory.
We agreed upon 4 parts from P category along with small spacer length of 6 between RBS and CDS (GFP). 6 parts of Q category with no additonal spacer. For each part RBS was calculated individually using Salis lab calculator.
20 July The assembled parts were send to anantha ma'am for screening to check if any unwanted restriction sites were created.
24 July We discussed again over the rationale and made several corrections.
27-29 July Sathvik, Sai Guha and Sarvesh present ADaPtat1on, ChassiDex and The Language project at all India iGEM meet.
30 July All team members come back. Burhan gives update on workplan of adaptation.

2 August: Promoter sequences are finalized and we place order.
7 August: iGEM wetlab work starts-1st plan is to clone platform construct.
10 August: Platform construction ligation was improper and gives band even at undesired region.
13 August: Troubleshooting of cloning of Platform construct in pBAV1k using Afl(II). All 20 promoters arrive.
17 August: We decide not to use platfrom construct. Instead PCR amplify the pBAV1k vector backbone using appropriate primers such that existing GFP can be excised out and Codon optimized GFP can be cloned.
20 August: We design appropriate primers for pBAV1k and placed the order. We also designed primers to clone promoters.
27 August: Primers arrive. We amplified the backbone and GFP gblocks(>12cycles). In all our PCR amplification experiments we used Phusion Polymerase HF.
28 August: After checking length on gel, restriction digestion was done and then we set it up for ligation (overnight).
29 August: A. baylyi was tranformed and we selected 5 colonies. We screened the colonies using Afl(II) as only insert had that unique site.
30 August: Colony1, 4 and 5 gave positive results and so they were selected.

12 September: Anantha ma'am allows Sankalpa to work in the lab. Her lab training starts.
13 September: Cloning of first 6 promoters in pSB1C3.
16 September: Cloning for first 6 promoters failed. Probably because amount of DNA was less.
17 September: PCR amplification of promoters and pSB1C3. This time we took only one promoter.
19 September: Colony PCR revealed successful transformation.
21 September: PCR amplification of pBAV1k such that T5 promoter remains unamplified. Other regions are amplified.
22 September: Cloning of next 4 promoters in pBAV1k and pSB1C3. Cloning of GFP into pSB1C3.
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25 September: Suspected contamination in our lab's plasmid isolation kit.
26 September: on plate of P2R pBAV1k, we did not get any colony. Rest all transformations were successful(ascertained using Colony PCR).
27 September: Attempt to clone next 15 promoters and mcherry in pSB1C3 vector for submission.