iGEM 2018 — Team IIT-Madras

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# Team:IIT-Madras/Notebook

iGEM Collaborations Page

Project

[Description](https://2018.igem.org/Team%3AIIT-Madras/Description)
[Design](https://2018.igem.org/Team%3AIIT-Madras/Design)
[Notebook](https://2018.igem.org/Team%3AIIT-Madras/Notebook)
[Results](https://2018.igem.org/Team%3AIIT-Madras/Results)
[Demonstrations](https://2018.igem.org/Team%3AIIT-Madras/Demonstrations)
[Improvements](https://2018.igem.org/Team%3AIIT-Madras/Improvements)
[InterLab](https://2018.igem.org/Team%3AIIT-Madras/InterLab)
[Safety](https://2018.igem.org/Team%3AIIT-Madras/Safety)

[Parts](https://2018.igem.org/Team%3AIIT-Madras/Parts)
[Software](https://2018.igem.org/Team%3AIIT-Madras/Software)

Human Practices

[Overview](https://2018.igem.org/Team%3AIIT-Madras/Human_Practices)
[Silver Human Practices](https://2018.igem.org/Team%3AIIT-Madras/Silver)
[Integrated Human Practices](https://2018.igem.org/Team%3AIIT-Madras/IntegratedHP)

Public Engagement

[Overview](https://2018.igem.org/Team%3AIIT-Madras/Public_Engagement)
[The Language Project](https://2018.igem.org/Team%3AIIT-Madras/TheLanguageProject)
[School Visits](https://2018.igem.org/Team%3AIIT-Madras/SchoolVisits)
[Research Magazine](https://2018.igem.org/Team%3AIIT-Madras/Synkranti)
[Workshop](https://2018.igem.org/Team%3AIIT-Madras/Workshops)
[SynBio Journal Club](https://2018.igem.org/Team%3AIIT-Madras/SynBioJournalClub)

The Team

[The Team](https://2018.igem.org/Team%3AIIT-Madras/Team)
[Attributions](https://2018.igem.org/Team%3AIIT-Madras/Attributions)
[Collaborations](https://2018.igem.org/Team%3AIIT-Madras/Collaborations)

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[Home](https://2018.igem.org/Team%3AIIT-Madras)
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[Description](https://2018.igem.org/Team%3AIIT-Madras/Description)
[Design](https://2018.igem.org/Team%3AIIT-Madras/Design)
[Notebook](https://2018.igem.org/Team%3AIIT-Madras/Notebook)
[Results](https://2018.igem.org/Team%3AIIT-Madras/Results)
[Demonstrations](https://2018.igem.org/Team%3AIIT-Madras/Demonstrations)
[Improvements](https://2018.igem.org/Team%3AIIT-Madras/Improvements)
[InterLab](https://2018.igem.org/Team%3AIIT-Madras/InterLab)
[Safety](https://2018.igem.org/Team%3AIIT-Madras/Safety)

[Parts](https://2018.igem.org/Team%3AIIT-Madras/Parts)
[Software](https://2018.igem.org/Team%3AIIT-Madras/Software)
Human Practices

[Overview](https://2018.igem.org/Team%3AIIT-Madras/Human_Practices)
[Silver Human Practices](https://2018.igem.org/Team%3AIIT-Madras/Silver)
Integrated Human Practices

Public Engagement

[Overview](https://2018.igem.org/Team%3AIIT-Madras/Public_Engagement)
[Language Project](https://2018.igem.org/Team%3AIIT-Madras/TheLanguageProject)
[School Visits](https://2018.igem.org/Team%3AIIT-Madras/SchoolVisits)
[Research Magazine](https://2018.igem.org/Team%3AIIT-Madras/Synkranti)
[Workshop](https://2018.igem.org/Team%3AIIT-Madras/Workshops)
[SynBio Journal Club](https://2018.igem.org/Team%3AIIT-Madras/SynBioJournalClub)

The Team

[The Team](https://2018.igem.org/Team%3AIIT-Madras/Team)
[Attributions](https://2018.igem.org/Team%3AIIT-Madras/Attributions)
[Collaborations](https://2018.igem.org/Team%3AIIT-Madras/Collaborations)

☰

[![iGEM IITM](https://static.igem.org/mediawiki/2018/e/ee/T--IIT-Madras--iitmlogo.png)](https://2018.igem.org/Team%3AIIT-Madras)

Team: IIT-Madras/Results

# Notebook

April 2018

**13 April:** We attempted making competent E. coli DH5alpha cells. We got the strain from Prof. Guhan Jayaraman's lab
**16 April:** We tried checking its competency by RFP transformation but did not get results.
**20 April:** We tried making competent cells again but we did not use liquid nitrogen for the flash freeze step. We used SOC media instead of LB this time.
**22 April:** We tried transformation but it failed in some plates. Much fewer colonies.
**26 April:**We met Guhan sir and Dr. Anantha Barathi Ma'am for guidance and help. Also, tried to analyze the feasibility of the project.

May 2018

**8 May:** Exams end and we discuss ideas with Anantha Ma'am (Instructor). One interesting study thought of was lignocellulosic mass conversion to other useful and economically highly valued products.
**8-10 May:** We do an extensive literature survey for this project and *Acinetobacter baylyi* ADP1 seems to be a good candidate.
**14 May:** We finalise work on lignin conversion to biofuels using *Acinetobacter baylyi* ADP1 as our project. We choose this organisms over others because it is non pathogenic and has amazing capabilities such as degrading aromatic compounds like caffeate, vaillate, etc.
Above all, it is naturally competent and Guhan sir's lab has expertise in working with this organism.
**16 May:** *Acinetobacter baylyi* has 6 copies of transposomal elements which were remove in 2017 to engineer a new strain 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 Prof. Barrick to send us the engineered strain.
**18 May:** We plan to compile all literature and decide on the 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 decide to shift the direction of our project towards expanding the toolbox of A. baylyi ADP1.
**23 May:** We finalise upon the things we want to try out: codon-optimized reporters and promoters. If time permits then terminators.
**25 May:** Anantha ma'am asks Sagar to place an order for CO-GFP and mCherry with GenScript.
Meanwhile we do a lit survey for various approaches for promoter design.
**29 May:** GenScript informs us that there is no reliable codon usage table available. They send us link to the 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:** A workplan was drawn up to start working on promoter Ddsign, codon usage table for 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 to work on the codon table of A. baylyi
Richa, Burhan and Sathvik get to work on promoter design.

June 2018

**4 June:** Codon generator was ready to be used on the ChassiDex website. We took the complete CDS annotation of A. baylyi and removed putative and hypothetical regions. We were left with 1194 CDS regions. We got the codon usage information. The data was then normalized and the 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 Mr. Anindhya of Medha Bioinformatics but all efforts to find conserved regions were fruitless.
Meanwhile, we codon optimize the GFP and mCherry using the codon table. We replaced the less frequently used codons with frequently used codons and screened for unwanted restriction sites. Then, we sent the sequence to Anantha ma'am for another checkup.
**8 June:** We used various other tools such as ClustalW, MEME suite, etc but could not find the consensus regions upstream of the genes that we had selected.
**10 June:** We discussed the generic nature of CUTE and planned to implement the same in the promoter sequences. We also discussed the nature of the pBAV1K vector that allowed it to replicate in so many host organisms.
**11 June:** We discussed and agreed upon making a T5 promoter-based library for our project.
**12 June:** GFP and mCherry designs were corrected wherever necessary. Planned to place order for the finalized sequences.
**13 June:** A literature survey of T5 promoter and promoter-based library was started.
**18 June:** The lab training period for Burhan and Sathvik was begun. Anantha Barathi ma'am, Sagar, Velvizhi Devi and Suchi Smita helped out with the training. Various basic molecular biology and microbiology techniques were taught.This training made us familiar with lab safety and lab rules and familiarized us with lab equipment. In parallel, we also explored the various methods and approaches for building a promoter library.
**19 June:** We placed the order for the codon-optimized GFP and mCherry to IDT. We finalized upon the parameters and started generating sequences.
**22-26 June:** Sathvik leaves for home, lab training continues. Burhan generates promoter library containing over 300 sequences of the same design. Meanwhile, since the project abstract deadline was approaching, we decide to christen it with a nice name. Sathvik names it 'ADaPtat1on- Expanding the toolkit of Acinetobacter baylyi'.
**29 June:** Burhan leaves and the lab training period subsequently ends.

July 2018

**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 various authors were taken into consideration and similar lines were followed to generate the promoter library.
**16 July** We have the promoter sequences from both categories, with the former being called the P the and the latter the Q series. We planned to add RBS' calculated using the RBS calculator tool from the Salis Lab website. Codon-optimized reporters arrive.
**18 July** We assembled the parts in-silico and then screened for unwanted restriction sites and agreed on the selection of parts from each category. We agreed upon 4 parts from P category along with small spacer length of 6 between RBS and CDS (GFP) and 6 parts of Q category with no additional spacer. For each part, the RBS was calculated individually using the Salis Lab calculator.
**20 July** The assembled parts were sent to Anantha Ma'am for screening to check if any unwanted restriction sites were created.
**24 July** We discussed the rationale again and made several corrections.
**27-29 July** Sathvik, Sai Guha and Sarvesh presented ADaPtat1on, ChassiDex and The Language Project at the All-India iGEM meet at the Indian Institute of Science, Bangalore.
**30 July** All team members return from vacations. Burhan gives an update on the work done on ADaPtat1on.

August 2018

**2 August:** Promoter sequences are finalized and the order for them is placed.
**7 August:** iGEM wetlab work starts- the first plan is to clone platform construct into the pBAV1K vector.
**10 August:** Platform construct ligation was improper and gave bands even at undesired region.
**13 August:** Troubleshooting of cloning of platform construct in pBAV1k using Afl(II) is done. All 20 promoters arrive.
**17 August:** We decide not to use the platfrom construct. Instead, we plan to 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 place the order. We also design primers to clone the promoters.
**27 August:** Primers arrive. We amplify the backbone and GFP gblocks(>12cycles). In all our PCR amplification experiments, we used the Phusion Polymerase HF.
**28 August:** After checking length on gel, restriction digestion was done and overnight ligation was set up.
**29 August:** A. baylyi was transformed and we selected 5 colonies. We screened the colonies using Afl(II) as only the insert had that unique site.
**30 August:** Colonies 1, 4 and 5 gave positive results and so they were selected.

September 2018

**12 September:** Sankalpa joins labwork.
**13 September:** Cloning of the first 6 promoters in pSB1C3.
**16 September:** Cloning of the first 6 promoters fails, robably due to the low amount of DNA used.
**17 September:** PCR amplification of promoters and pSB1C3 is done. This time, we use only one promoter.
**19 September:** Colony PCR reveals successful transformation.
**21 September:** PCR amplification of pBAV1K is done such that T5 promoter remains unamplified. Other regions are amplified.
**22 September:** Cloning of the next 4 promoters in pBAV1k and pSB1C3 is done. Cloning of GFP into pSB1C3 is also carried out.
**25 September:** Contamination is suspected in our lab's plasmid isolation kit.
**26 September:** We did not get any colony on the P2R pBAV1K plate. The remaining transformations were all successful (as ascertained using colony PCR).
**27 September:** We attempt to clone the next 15 promoters and mCherry in the pSB1C3 vector for BioBrick submission.

October 2018

**1 October:** Fluorometry of CO-GFP was unsuccesful.
**2 October:** Set up a gradient PCR of the promoters
**3 October:** Amplification of promoters and cloning of promoters into pBAV1K.
**4 October:** Cloning of promoters into pBAV1K and transformation into *A. baylyi*
**5 October:** Transformation of promoters into *A. baylyi*
**6 October:** Plasmid isolation of pSB1C3 containing promoters from *E. coli*.
**7 October:** Fluorometry of CO-GFP and first set of promoters
**8 October:** Fluorometry of second set of promoters.


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# Team:IIT-Madras/Experiments

iGEM Collaborations Page

Project

[Description](https://2018.igem.org/Team%3AIIT-Madras/Description)
[Design](https://2018.igem.org/Team%3AIIT-Madras/Design)
[Notebook](https://2018.igem.org/Team%3AIIT-Madras/Notebook)
[Results](https://2018.igem.org/Team%3AIIT-Madras/Results)
[Demonstrations](https://2018.igem.org/Team%3AIIT-Madras/Demonstrations)
[Improvements](https://2018.igem.org/Team%3AIIT-Madras/Improvements)
[InterLab](https://2018.igem.org/Team%3AIIT-Madras/InterLab)
[Safety](https://2018.igem.org/Team%3AIIT-Madras/Safety)

[Parts](https://2018.igem.org/Team%3AIIT-Madras/Parts)
[Software](https://2018.igem.org/Team%3AIIT-Madras/Software)

Human Practices

[Overview](https://2018.igem.org/Team%3AIIT-Madras/Human_Practices)
[Silver Human Practices](https://2018.igem.org/Team%3AIIT-Madras/Silver)
[Integrated Human Practices](https://2018.igem.org/Team%3AIIT-Madras/IntegratedHP)

Public Engagement

[Overview](https://2018.igem.org/Team%3AIIT-Madras/Public_Engagement)
[The Language Project](https://2018.igem.org/Team%3AIIT-Madras/TheLanguageProject)
[School Visits](https://2018.igem.org/Team%3AIIT-Madras/SchoolVisits)
[Research Magazine](https://2018.igem.org/Team%3AIIT-Madras/Synkranti)
[Workshop](https://2018.igem.org/Team%3AIIT-Madras/Workshops)
[SynBio Journal Club](https://2018.igem.org/Team%3AIIT-Madras/SynBioJournalClub)

The Team

[The Team](https://2018.igem.org/Team%3AIIT-Madras/Team)
[Attributions](https://2018.igem.org/Team%3AIIT-Madras/Attributions)
[Collaborations](https://2018.igem.org/Team%3AIIT-Madras/Collaborations)

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[Home](https://2018.igem.org/Team%3AIIT-Madras)
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[Description](https://2018.igem.org/Team%3AIIT-Madras/Description)
[Design](https://2018.igem.org/Team%3AIIT-Madras/Design)
[Notebook](https://2018.igem.org/Team%3AIIT-Madras/Notebook)
[Results](https://2018.igem.org/Team%3AIIT-Madras/Results)
[Demonstrations](https://2018.igem.org/Team%3AIIT-Madras/Demonstrations)
[Improvements](https://2018.igem.org/Team%3AIIT-Madras/Improvements)
[InterLab](https://2018.igem.org/Team%3AIIT-Madras/InterLab)
[Safety](https://2018.igem.org/Team%3AIIT-Madras/Safety)

[Parts](https://2018.igem.org/Team%3AIIT-Madras/Parts)
[Software](https://2018.igem.org/Team%3AIIT-Madras/Software)
Human Practices

[Overview](https://2018.igem.org/Team%3AIIT-Madras/Human_Practices)
[Silver Human Practices](https://2018.igem.org/Team%3AIIT-Madras/Silver)
Integrated Human Practices

Public Engagement

[Overview](https://2018.igem.org/Team%3AIIT-Madras/Public_Engagement)
[Language Project](https://2018.igem.org/Team%3AIIT-Madras/TheLanguageProject)
[School Visits](https://2018.igem.org/Team%3AIIT-Madras/SchoolVisits)
[Research Magazine](https://2018.igem.org/Team%3AIIT-Madras/Synkranti)
[Workshop](https://2018.igem.org/Team%3AIIT-Madras/Workshops)
[SynBio Journal Club](https://2018.igem.org/Team%3AIIT-Madras/SynBioJournalClub)

The Team

[The Team](https://2018.igem.org/Team%3AIIT-Madras/Team)
[Attributions](https://2018.igem.org/Team%3AIIT-Madras/Attributions)
[Collaborations](https://2018.igem.org/Team%3AIIT-Madras/Collaborations)

☰

[![iGEM IITM](https://static.igem.org/mediawiki/2018/e/ee/T--IIT-Madras--iitmlogo.png)](https://2018.igem.org/Team%3AIIT-Madras)

Team: IIT-Madras/Results

# Lorem Ipsum

## Protocols

**Qiagen Plasmid Mini Kit**

1. Pick a single colony from a freshly streaked selective plate and inoculate a starter culture of 2–5 ml LB medium containing the appropriate selective antibiotic. Incubate for approximately 8 h at 37°C with vigorous shaking (approx. 300 rpm). Use a tube or flask with a volume of at least 4 times the volume of the culture.
2. Dilute the starter culture 1/500 to 1/1000 into 3 ml selective LB medium. Grow at 37°C for 12–16 h with vigorous shaking (approx. 300 rpm). Use a flask or vessel with a volume of at least 4 times the volume of the culture. The culture should reach a cell density of approximately 3–4 x 109 cells per milliliter, which typically corresponds to a pellet wet weight of approximately 3 g/liter medium.
3. Harvest the bacterial cells by centrifugation at 6000 x g for 15 min at 4°C. If you wish to stop the protocol and continue later, freeze the cell pellets at –20°C.
4. Resuspend the bacterial pellet in 0.3 ml of Buffer P1. Ensure that RNase A has been added to Buffer P1. If LyseBlue reagent has been added to Buffer P1, vigorously shake the buffer bottle before use to ensure LyseBlue particles are completely resuspended. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.
5. Add 0.3 ml of Buffer P2, mix thoroughly by vigorously inverting the sealed tube 4–6 times, and incubate at room temperature (15–25°C) for 5 min. Do not vortex, as this will result in shearing of genomic DNA. The lysate should appear viscous. Do not allow the lysis reaction to proceed for more than 5 min. After use, the bottle containing Buffer P2 should be closed immediately to avoid acidification from CO2 in the air. If LyseBlue has been added to Buffer P1, the cell suspension will turn blue after addition of Buffer P2. Mixing should result in a homogeneously colored suspension. If the suspension contains localized colorless regions or if brownish cell clumps are still visible, continue mixing the solution until a homogeneously colored suspension is achieved.
6. Add 0.3 ml of chilled Buffer P3, mix immediately and thoroughly by vigorously inverting 4–6 times, and incubate on ice for 5 min. Precipitation is enhanced by using chilled Buffer P3 and incubating on ice. After addition of Buffer P3, a fluffy white material forms and the lysate becomes less viscous. The precipitated material contains genomic DNA, proteins, cell debris, and KDS. The lysate should be mixed thoroughly to ensure even potassium dodecyl sulphate precipitation. If the mixture still appears viscous, more mixing is required to completely neutralize the solution. If LyseBlue reagent has been used, the suspension should be mixed until all trace of blue has gone and the suspension is colorless. A homogeneous colorless suspension indicates that the SDS has been effectively precipitated.
7. Centrifuge at maximum speed in a microcentrifuge for 10 min. Remove supernatant containing plasmid DNA promptly. Before loading the centrifuge, the sample should be mixed again. Centrifugation should be performed at maximum speed in 1.5 ml or 2 ml microcentrifuge tubes (e.g., 10,000–13,000 rpm in a microcentrifuge). Maximum speed corresponds to 14,000–18,000 x g for most microcentrifuges. After centrifugation, the supernatant should be clear. If the supernatant is not clear, a second,shorter centrifugation should be carried out to avoid applying any suspended or particulate material to the column. Suspended material (which causes the sample to appear turbid) will clog the column and reduce or eliminate flow. Optional: Remove a 50 µl sample from the cleared lysate and save it for an analytical gel (sample 1).
8. Equilibrate a QIAGEN-tip 20 by applying 1 ml Buffer QBT, and allow the column to empty by gravity flow. Place QIAGEN-tips into a QIArack over the waste tray or use the tip holders provided with each kit (see “Setup of QIAGEN-tips” page 13). Flow of buffer will begin automatically by reduction in surface tension due to the presence of detergent in the equilibration buffer. Allow the QIAGEN-tip to drain completely. QIAGEN-tips can be left unattended, since the flow of buffer will stop when the meniscus reaches the upper frit in the column.
9. Apply the supernatant from step 7 to the QIAGEN-tip 20 and allow it to enter the resin by gravity flow. The supernatant should be loaded onto the QIAGEN-tip promptly. If it is left too long and becomes cloudy due to further precipitation of protein, it must be centrifuged again before loading to prevent clogging of the QIAGEN-tip. Optional: Remove a 50 µl sample of the flow-through and save for an analytical gel (sample 2).
10. Wash the QIAGEN-tip 20 with 2 x 2 ml Buffer QC. Allow Buffer QC to move through the QIAGEN-tip by gravity flow. Optional: Remove a 220 µl sample of the combined wash fractions and save for an analytical gel (sample 3).
11. Elute DNA with 0.8 ml Buffer QF. Collect the eluate in a 1.5 ml or 2 ml microcentrifuge tubes (not supplied). Note: For constructs larger than 45–50 kb, prewarming the elution buffer to 65°C may help to increase yield. Optional: Remove a 45 µl sample of the eluate and save for an analytical gel (sample 4).
12. Precipitate DNA by adding 0.7 volumes (0.56 ml per 0.8 ml of elution volume) of room-temperature isopropanol to the eluted DNA. Mix and centrifuge immediately at ≥15,000 x g rpm for 30 min in a microcentrifuge. Carefully decant the supernatant. All solutions should be at room temperature to minimize salt precipitation. Isopropanol pellets have a glassy appearance and may be more difficult to see than the fluffy, salt-containing pellets that result from ethanol precipitation. Marking the outside of the tube before centrifugation allows the pellet to be easily located. Isopropanol pellets are also more loosely attached to the side of the tube, and care should be taken when removing the supernatant.
13. Wash DNA pellet with 1 ml of 70% ethanol and centrifuge at 15,000 x g for 10 min. Carefully decant the supernatant without disturbing the pellet. The 70% ethanol removes precipitated salt and replaces isopropanol with the more volatile ethanol, making the DNA easier to redissolve.
14. Air-dry the pellet for 5–10 min, and redissolve the DNA in a suitable volume of buffer (e.g., TE buffer, pH 8.0, or 10mM Tris·Cl, pH 8.5) Redissolve the DNA pellet by rinsing the walls to recover the DNA. Pipetting the DNA up and down to promote resuspension may cause shearing and should be avoided. Overdrying the pellet will make the DNA difficult to redissolve. DNA dissolves best under slightly alkaline conditions; it does not easily dissolve in acidic buffers.

**Lorem Ipsum**

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### References

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[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:IIT-Madras/Results

iGEM Collaborations Page

Project

[Description](https://2018.igem.org/Team%3AIIT-Madras/Description)
[Design](https://2018.igem.org/Team%3AIIT-Madras/Design)
[Notebook](https://2018.igem.org/Team%3AIIT-Madras/Notebook)
[Results](https://2018.igem.org/Team%3AIIT-Madras/Results)
[Demonstrations](https://2018.igem.org/Team%3AIIT-Madras/Demonstrations)
[Improvements](https://2018.igem.org/Team%3AIIT-Madras/Improvements)
[InterLab](https://2018.igem.org/Team%3AIIT-Madras/InterLab)
[Safety](https://2018.igem.org/Team%3AIIT-Madras/Safety)

[Parts](https://2018.igem.org/Team%3AIIT-Madras/Parts)
[Software](https://2018.igem.org/Team%3AIIT-Madras/Software)

Human Practices

[Overview](https://2018.igem.org/Team%3AIIT-Madras/Human_Practices)
[Silver Human Practices](https://2018.igem.org/Team%3AIIT-Madras/Silver)
[Integrated Human Practices](https://2018.igem.org/Team%3AIIT-Madras/IntegratedHP)

Public Engagement

[Overview](https://2018.igem.org/Team%3AIIT-Madras/Public_Engagement)
[The Language Project](https://2018.igem.org/Team%3AIIT-Madras/TheLanguageProject)
[School Visits](https://2018.igem.org/Team%3AIIT-Madras/SchoolVisits)
[Research Magazine](https://2018.igem.org/Team%3AIIT-Madras/Synkranti)
[Workshop](https://2018.igem.org/Team%3AIIT-Madras/Workshops)
[SynBio Journal Club](https://2018.igem.org/Team%3AIIT-Madras/SynBioJournalClub)

The Team

[The Team](https://2018.igem.org/Team%3AIIT-Madras/Team)
[Attributions](https://2018.igem.org/Team%3AIIT-Madras/Attributions)
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Team: IIT-Madras/Results

# Results

##

**Promoter results:**

From the initial set of 20 promoters, we got successful cloning results for 15 promoters. We performed fluorometry experiments and obtained 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 the control, which we got from Addgene(https://www.addgene.org/267), and the wild-type as the negative control.

All promoters were cloned into a pBAV1k vector backbone with GFP. Fluorometry studies were then carried out with each clone.
From these 15 promoters, we got a spectrum of results showing varying expression. The R-Series promoters (promoters having the Salis lab calculated RBS) mostly exhibited better expression rates as compared to those of the S-series.

For each promoter, we had two biological replicates and three technical replicates. The errors and differences in each of these are depicted in the error bars.

Promoters P8R (BBa\_K2857116), P8S (BBa\_K2857118), P9R (BBa\_K2857119), P1R (BBa\_K2857111), P1S (BBa\_K2857101), P8S (BBa\_K2857012), and P3R (BBa\_K2857113) showed higher expression than the T5 promoter.

![](https://static.igem.org/mediawiki/parts/6/6a/T--IIT-Madras-Promotersjgp.png)

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) showed lower expression than the T5 promoter.

**P8S and T5 promoter:**
P8S showed tremendously high expression of GFP, 75-fold greater than the control.
![](https://static.igem.org/mediawiki/parts/5/5e/T--IIT-Madras-P8S.png)

#### **Conclusion:**

* We have been successful in obtaining a spectrum of promoters which is desirable while generating a synthetic promoter library.
* We have obtained promoters which show higher expression as well as ones showing lower expression than the T5 promoter.
* We have designed a promoter P8S which shows about 75 fold higher expression than T5 promoter

**Codon optimized GFP and Codon usage table results:**

From the *Acinetobacter baylyi* ADP1 genome we obtained 1194 genes after removing hypothetical and putative sequences from the coding sequences annotation available from NCBI. The Codon Usage Table Enumerator (CUTE) tool from ChassiDex was 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*

![](https://static.igem.org/mediawiki/2018/a/a5/T--IIT-Madras--codonFig1.png)

The table attached below lists the percentage distribution of different codons amongst the analyzed genes.

![](https://static.igem.org/mediawiki/2018/1/15/T--IIT-Madras--Fig2.png)

The table attached below gives the probability of a particular amino acid being coded for by a particular codon.

![](https://static.igem.org/mediawiki/2018/5/55/T--IIT-Madras--Fig3.png)

These tables were then used to codon-optimize reporter proteins - GFP and mCherry - using the sequences available from NCBI. Codon-optimized GFP was restriction-cloned into a pBAV1K vector and transformed into *A. baylyi*. It showed promising results as illustrated below. The codon-optimized reporter protein showed considerably higher expression of fluorescence than the control.

The magnitude of the fluorescence readings on the y-axis is 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 column shows fluorometry data for *Acinetobacter baylyi* ADP1 with the codon-optimized GFP in a pBAV1k vector under the T5 promoter. The error bars were calculated by taking into account two biological replicates and three technical replicates of each biological replicate.

![](https://static.igem.org/mediawiki/parts/1/1c/T--IIT-Madras-GFP-characterization.png)

#### **Conclusion:**

* From the fluorometry results we can note that codon-optimized GFP shows higher expression in comparison to the control. This clearly demonstrates that the expression of codon-optimized GFP is greater than that of the non-codon optimized GFP.
* This suggests that codon optimization has been successful.
* This further indicates that the CUTE was successful in generating a valid codon-usage table.