# Team:IISER-Tirupati India/Contribution

Ovi-Cloak

## New Parts:

### Promoters:

In order to achieve robustness in the system, it is necessary to have a library of promoters with a wide range of transcription rates. One such library of synthetic promoters from Liu et al. (2018) consisted of 214 synthetic promoters with consensus sequence as shown below [1]:

Fig. 1 SP Backbone

All these promoters are constitutive hence can be used for general protein production. From this library we used SP126, SP146 and SP200 having relative activity with respect to P43 as follows:

 Promotor Sequence 5' → 3' Relative activity wrt P43- GFP (%) Standard deviation SP126 AAAAATTATAAAAATGTGTTGACAAAGGGGGTCCTGTATGTTATAATAGCTT 29.07 0.23 SP146 AAAAATAACAAAAACGTGTTGACAATAAAGATTAACCGTGATATAATTAAAT 40.39 0.69 SP200 AAAAATTAGAAAAATGTGTTGACACTCGGACGAAACAATGGTATAATGGCAA 76.82 0.9

### P22 Operator Library:

P22 c2 repressor (BBa_K3889020, BBa_C0053) binds to the binding site (operator site) as a dimer. This inhibits the enzymes from transcribing the genes on whose promoter this operator site is fused. Hence this could be used with any promoter to form a repressible system. Different binding affinities of a repressor provide a variable system that can be used for different expression levels of the target, thereby enabling it in a variety of systems [2]. Optimization and tweaking of a system can be done by varying the operator sites as well.

 Part Name Sequence Rel KD K D (in M) P22 binding site A ATTTAAGATATCTTAAAT 1 1.6 × 10−8 P22 binding site B AATTAAGATATCTTAATT 1.8 2.88 × 10-8 P22 binding site C ATTTAAGAATTCTTAAAT 2 3.2 × 10−8 P22 binding site D AGTTAAGATATCTTAACT 2.6 4.16 × 10−8 P22 binding site E ATTAAAGATATCTTTAAT 3.8 6.08 × 10−8 P22 binding site F ACTTAAGATATCTTAAGT 4.3 6.88 × 10−8 P22 binding site G ATTCAAGATATCTTGAAT 5 8.0 × 10−8 P22 binding site H ATTGAAGATATCTTCAAT 7.6 1.216 × 10−7 P22 binding site I ATTTAAGAGCTCTTAAAT 10 1.6 × 10−7 P22 binding site J ATTTAAGACGTCTTAAAT 10 1.6 × 10−7 P22 binding site K ATTTACGATATCGTAAAT 30 4.8 × 10−7 P22 binding site L ATTTAAAATATTTTAAAT 55 8.8 × 10−7

### Coding sequences:

SRTF1 or steroid responsive transcription factor 1 can negatively regulate any promoter activity if its binding site is fused with the gene's promoter. SRTF1 binds to its binding site(BBa_K3889030) as done in BBa_K3889150. Presence of progesterone causes unbinding of SRTF1 thereby releasing it from the DNA, inducing the target gene.Thus,progesterone acts as an inducer and can be used in a progesterone inducible system by other teams as well [3].

### Device:

Terminator checking device (BBa_K3889140): In order to check terminator efficiency a simple reference circuit was used similar to what used by Gale et al. (2021) [4] as shown below:

Now, spacer can be replaced with any terminator to see the expression of sfGFP and mCherry.

Formulae for terminator efficiency [4] :

$TE_{Device} = \frac{mCherry_{0}}{sfGFP_{0}}$

where,

$mCherry_{0} \rightarrow$ mCherry produced by device without terminator

$sfGFP_{0} \rightarrow$ sfGFP produced by device without terminator

Using the device/reference without any changes, $TE_{Device}$ can be calculated which gives the expression of $mCherry$ in absence of a terminator.

$$\tag{2}TE=100-\left[\left(\frac{mCherry}{sfGPF}\right)\times\left(\frac{1}{TE_{Device}}\right)\times100\right]$$

where,
$mCherry \rightarrow$ mCherry produced by device with the terminator that needs to checked

$sfGFP \rightarrow$ sfGFP produced by device with the terminator that needs to checked

### Modifications in the old parts:

 Old part New part Name Modifications made BBa_K143036 BBa_K3889092 XylR Removed Dual stop codons BBa_C0053 BBa_K3889020 P22 c2 repressor Removed Sap1 Recognition Site, LVA tag and Barcodes BBa_K1442039 BBa_K3889069 P2A Peptide Linker PTV BsmBI recognition site removed for Golden Gate compatibility BBa_K3507002 BBa_K3889025 YqcG toxin Mutated Xho1, HindIII and Bsa1 sites to make assembly compatible part BBa_K3507003 BBa_K3889093 YqcF antitoxin Mutated BgIII site BBa_K143037 BBa_K3889026 YtvA Mutated Bsa1 and HindIII Sites BBa_K3519004 BBa_K3889027 bovine pancreatic DNase 1 Mutated HindIII Site BBa_K2333011 BBa_K3889028 mf-Lon protease Mutated multiple RE sites BBa_I746916 BBa_K3889002 sfGFP Mutated Kpn1 site and stop codon

### REFERENCES

1. Liu, D., Mao, Z., Guo, J., Wei, L., Ma, H., Tang, Y., Chen, T., Wang, Z., & Zhao, X. (2018). Construction, Model-Based Analysis, and Characterization of a Promoter Library for Fine-Tuned Gene Expression in Bacillus subtilis. ACS Synthetic Biology, 7(7), 1785–1797. https://doi.org/10.1021/acssynbio.8b00115
2. Watkins, D., Hsiao, C., Woods, K. K., Koudelka, G. B., & Williams, L. D. (2008). P22 c2 Repressor− Operator Complex:  Mechanisms of Direct and Indirect Readout. Biochemistry, 47(8), 2325–2338. https://doi.org/10.1021/bi701826f
3. Baer, R. Cooper (2020). Discovery, characterization, and ligand specificity engineering of a novel bacterial transcription factor inducible by progesterone Boston University School of Medicine, 801 Massachusetts Avenue Suite 400 Boston, MA 02118 Retrieved from: https://hdl.handle.net/2144/41109
4. Gale, G. A. R., Wang, B., & McCormick, A. J. (2021). Evaluation and Comparison of the Efficiency of Transcription Terminators in Different Cyanobacterial Species. Frontiers in Microbiology, 11. https://doi.org/10.3389/fmicb.2020.624011

## Gene Gala

We held a Mini-Summer school in collaboration with the iGEM 2021 team of IISER Kolkata. It was a 5-day Mini-Summer School for Girl students studying in 12th Standards of the schools under the Directorate of Education, GNCT Delhi. As part of the summer school, the two teams together prepared a 5-days lesson plan, two quiz sessions, and a day-to-day handbook made for reference for the students. We would like to present these resources as a contribution to iGEM.

Future iGEM teams can use them directly for conducting similar programs in their regions/countries to the relevant audiences giving proper attributions to both the contributing teams. These resources will be extremely useful for teams who are preparing for similar education events. Conducting classes for five days enriched with activities and quiz sessions can be a daunting task for teams. The lesson plan provided here was able to keep the students engaged throughout the five days and it was easy for the team members to present as well. These content handbooks, lesson plans, and quizzes will come in handy for future iGEM teams to prepare for such an event and take their public engagement to the next level

The content is relevant for introducing high school seniors to Synthetic Biology while giving them a holistic and application-based view of the biology courses taught at the high school level.