iGEM 2020 — Team CU-Boulder

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# Team:CU-Boulder/Notebook

# Notebook

![](https://lh3.googleusercontent.com/pS2pcgaknOs1AeQ7YXCfWtp8R02F3MOnjnpehtILesWpxlj359IcbG12PXwc3m7Lu6v677xS4IoKmy-KDFxSRQRim1FcqoAv9OP-JqYYaYwdpV-ROlLYsJsuFUnVarPzG5Em0rwcU3dN4IiUzlpVEF7UKueZlfEjyWeWyWbrNWl05TpKVXNH_Ces5GE11nfjT6Ceh-cMLA4rWVXg7iyXMoxHlzXQj8JCIFj9lFOuC2RA48gOgrTCQZP_1N_tUYleMyOTL0pWh2pAdc_SeLMJ0PGFVyYF9DlfZmvNkzq5-G5RtPkZqkrzcO6iK9w07RgfziNLcYnBkNqxlNzpN-h3tGhq_t4ZysiMBTcT0pCHRFLK_Cz4M21ALK8ZNUUghpVYObRuZLsReBu_WUKCdP6ekvR5oIS9UhlCwkA2mOcrU8NYKkibFb0UOiQZJuWJupEyEwGOjLhCweGGgJqGVoar68WyDTq4OGh1bVBjWFo1PoHxY-pTpLZqu5uW0NRvE8Tuvo2GxvRMSzVTVZriWak7EmHgDG3eAAIXHOiJlrLbrstCxkFZCOa9gaDTmgnRR1rP6aYXpH9owDJFo2UHfab1bAyQo9wyH5m9-1dPLY89SOAHdDRRbxurGOpKA8nDf33i1cQcDYDr1cFIyIM5v9-SGkfX4MXfcC2LM9DyGX5Nddiji8_7VmsTeEZK-ho=w456-h360-no?authuser=2)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:CU-Boulder/Engineering

# Engineering and Modeling

We succeeded in designing the model of nitrogenase connected to the superoxide dismutase complex with a leucine zipper using PyMol. The screenshot of this is shown below.

![](https://lh3.googleusercontent.com/MBRo5Isw7rHoKLcCI4IymxryqAKKEdc-FvEKIV16OaH2oGkoj-Q1U_FKhg1Fm0G2hlBBK5tjANhr0xD2T0dX21MA6I6Ws6SR1ulZ7F87ig-T-L6FWIWij6Wgsm_NaQ57vO6pyNx07GQZFeVLUVv9TU-MSL_Lp7DHv5CjT4cshMfY9la6mwqZi0ckDrIU7nkqrI7Sit7ME_ZYFUSnPYsdgepJYuXNoSMkbWnoHtxSS3Ja9pN7gWwp2Qjwwz0e1zoChf1LRYAEaxj9kFIAIssVWGKKIBq_K72NisU8s1qUm1akPwPuX9y3EO4axaNpSBvS_J_cd35qEHkJLNvQDWCkLT8JPcf9v4mybLUTrwoPP1TSdJHMkna1K18I_6yDtanzLJJB33tLA8nPIEhVf_Mv2RNFw9jt0PQlkZ2k-eMj6tDMx8mFsz5fZTaUIFt_K5LmGrneRSxvhyEVc2tGXVDxvOVkI-vgI2DI7w6hM3aoeHMtKVCdax26pjwIbeCVgZ_BCL3L7sFs2pD8K72WaUhSPzo4A23UD426JieWGcFzckUwwLnEF_KkQ7M7e7fPML600BqN5sQmblULr84wrBMmXO_0n35Ikcc_H0omHLYBFbLE_JuJfqbhKAjDybl3NnYksNKfM9RHbXPUjIWgdAqmIt_mDOTC9OhPCFzVEeeSrzgBrBEKqF7PNPcUYXo=w521-h454-no?authuser=2)

Additionally, we were able to use PyRosetta to show the energy of different states of our engineered nitrogenase connected with superoxide dismutase. Below is an example output of the score for a biomolecule. The score in PyRosetta corresponds to the energy of a given molecule.

![](https://lh3.googleusercontent.com/WObF5T1H2R6nhCCT3XiDwjaWrvYoqo3yfhXi9djCsJwH4LwKJQDTHWmBwHiaIo-f835goEIZHGIcwzQ3ayaQs3AMWZj8m3EISo2Eaf36vnbccca1QrUH3BNMEMWs3cLEXnlfpkUDW4g7S-YZEIAZkL4RLsKBBWcV__TMJHK6fh8yAE2PZTXGvC9fjq2dqsU3lorri0CVG_fszAoupMAv5VY4Pit7NET37agrJAoUYbUEXRwiHjHusHG0LiG5Gouopi0JnoFhvELhsvczw_wxwStpaJfktGsrIGKKbGCpY1FWwGuHG0lVbXu-IflfKcRSX0auMeIlVFpY7W8uczf0F2yTlW-y5XwZMWuZiAuaobt70LsNeflg7GRm89OVHcVUR-H-GUpty_dhuWaU0Za610HxQKNb-retlzeya39cO97-s7gSYBip6hKWlg0d2JMHaamxFnUlqAvSljJH3DUtBjVU-5iXe5Ed1AsQhaAJUVdp1-R-JwLbAVoSmxcht-qe7956OrM6wHDUVoEayGKoSRoGp8oO2iTQMkRO0vASv5SsN_ORDMPRX49m85QXkWY06u9gLWNjjEJtOjeYex82won5PF4bUbm1fF_VecEwiggA6nC-fDNy5_1QOdli4LwEgxpt3VXVebhJAkuAuK_YlE81BEQ78lr6-qC3h222of1osOC-2re72hIjqPU=w968-h642-no?authuser=2)

Below are some of the code snippets of our PyRosetta modeling. The first image is the code to calculate the initial energy of our biomolecule. The second is an example setup of getting the actual score or energy and the third image is the code to execute an example run.

![](https://lh3.googleusercontent.com/FXuReY2js0iXnem97khtE-CyKjL2i43SgBcX0RVQmuhq0rFHVzWlZIVhNWzs5efxLHvlDcPCmqa772m_cry2PQOQ8nTArrl6dkd2XIXDark-nun3iRSI8xsAC5icoglbmgGStAv62UtLcAlYb65t_Ks0gy4I76IDvXm_SXqWWSebSfSbptn0dN_p_XkcEqTmOyXd_nO2uMr0xHCCqhMLDscEyanvtBpu14swhk5rTb4jnJWkwkbH-s2jcskQ5N2ZCZDAcyOKcDf-OdAYsx7FO6UTKt5QFRueE_45TQLPKv05LvejOAJPMonOR88g7EsGlGafWVLuU3P5uH4svIAphse4c9JhaWifVhAs-FPGxaS-vskZOZDtaYPw8e2IgvnuEqb6DGCMBKNYgs_-uSqulHMLI-PsLL6h5ULoLRNvZjR1DkGB4P7O8eusPXI1V_eX3gWDA-O3Qbthpqlc9BrOjGLfZnsUmjdhAUQGc5Jsg06hvWHXMkxrs9en1iZtH0Ek4cihOVyL4-jT9J9QjzT5Ew_MmK3lfYEBhDElfaYELZF3N8u6BgwVqrT-0NcCs_enFCRG8JGxmcbm1WD9w6GwJsSGW1GjGT2AFEzQ6TFwckovOf7omCZdaTe9IN2_V55WvYIinl-97MzPe15fanlqI4LNQmL1i15AQ4anN-gZSNuCI8Stkdwm2hJCdRE=w1491-h213-no?authuser=2)
![](https://lh3.googleusercontent.com/jyxlt_gh7OdB9wOckNJZzQEX1lHX-z2ijzAY-kpBZeCb-wh366tSyCkTKfN0yjditkyPAfG_U00B966CYyYEH-DHl4PnslwKQ_dudJ8msjaOMJsjvPEQy4SV2bE22FZVUZL3wHIsqEG-DC0UL8bhm3Vz1-AvcS-8mkzA94XMDPHQIg1wGL2cRlue18U86HzXKzRE-roTOpT1Cfq3F93-kTL_LBc-V5739PF0ALKpf_YfqPK1lU64xk2RunQAMJi6l-tZCdiIFmVeljjghlUqm1ve9vlvkWa_plBCemllWtpHloJfTKqnC_bbMtgypFnkiFhy_F-pyGLh1MZQTYuLIWT84HoNVj8amb1l8hCUBBjYYbY2Eb3ZSgaQ8yWSogW0-DEYU7o1gyA4UtZgviLEo8-aSplRV91UqaeD5ag0l0yQjMM-pJEGAY2kdrJ_H1ChI1EPeKxp8JefPfjGav0NT5G_r0ggCXKFjirEKideeCAXZ79FRZP207DTlJe0AG9rKlO3pofjHLIpLjctZLrzPzxVpFKR8qjChCQW_mqTLkd69E9YHIrdN1jXrC8l5tuef20GojsDbf0JwCYkyDSKXYTfen1FfFYSZTcX5YltQgjJ39msnDE5pQufohrQDXZGNbLGb7V9812BsdQWJjnkvcQwl6D4D8RmHLCE4UsM_jfjWJlohZZ5no55XxA=w737-h534-no?authuser=2)
![](https://lh3.googleusercontent.com/USAMSiO-d3AeCo5oVYPDh5p25vbLtVxBLIrFjk9YX1KhjVN9alZ5E2kXrqWx5AGxWdAt2_9HCPGCMROyWjK_4dHjthW6vbFGkaB5S6NQ939aVNkPBnbZlJdaFFpexa822OUy0gSEz8q9AZgbHBEmW1ci_uLWZw3DbTTGscWNMkKgfagDosAusH2BQMK0UKlrMsl4BN2dS20nkvKT9IeU9VhgsmHeWrNdSg2qgw1_pJWbaMYfuJWtpORuMQl-RRXWdmM1uFWDYP7GlyiyzOKiBr9BLStwOkkyfrB65QCrInNOrnmI1ea7pmDCMwG2-dzOOo3zQrGm3_jjyQrAyY9KJWNinN_HzH14eu6PyZpM_7TvtgXYqVqjAhHe86pkJ_y3c0YESGURT99UAYZNiAw2UbY9eeDbkrHv688wnqUw9QH93oMsv9jW73v_RY5sWTBFgRtXnKFVEPGSd47A0yzXdT7c4K_vqfPoavlr09vWUy6WVXUd1evccedvok2Iu3DSZT31ge2Fxa8QAc3GavCTcffHb5hq137IlBlqhIuyE2sQfr2TjY77Qsxw7PM8frn0QYrpLril3OfUNbrCGz-14M6LZ9xstM3LqV-b0xAKU3Pj4KrHBYu4v1pjDN7IQXPEHammA13kQ8WaA7gUAJp-iOHfZjX0Z5f5SwjB39MKEJuLEacwg7TYi3I9RCs=w514-h240-no?authuser=2)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:CU-Boulder/Experiments

# Experiments/Results

Given that we are on the Two-Year Track, all of our experiments revolve around the modeling of our system.

PYMOL:
We used Pymol to visualize and connect our potential models, as seen in the image below:

![](https://lh3.googleusercontent.com/7bpuI7knqyv0KqQo0cP28Auib1Xemr5QoQpABIK-N4n1H5z8zjS2dkyKGeh_DOa2lgCuJIeZlFew89Hv7IVGsd8m7M_lNhgm9Ol_9zzUN2QuJLlH07qn60-YPNGIvWQ9TgLJ0UykB861SwMV6cIXxhvqeMJouA1W8AfbJLoWFfMuxg2T-2D-TAxx-w7ZxCwtgzcWzrEPf4YlbQBZQ3Fd4bj0eIMeR8Wr_IHLCQ5wyxu_mFiC7C6CZgzBsCS6alRJCOkjmYmFTtUjHYcqbUAW3PhQf9LioiAAhog43KxAp8zfevm6TectLMzx1tC17Rr6xxrP9m8jN67f5PjwsBgcDhsMXMVhVWQdu8Rfy6J-Sob3lBEDEjq5K1iXnpq-wJVdIzix8rr2jOc6b6dNMGwN3kUtcaO5rqZd-b--a0ZVYTbq24RPFAjGaxsDfsOmfUWSfdi7naAZzXwGqxqOEkI61kHQrahpE4gN0R4HE8Gr4dc6ML-pY446x6x90jizAYA_E0Z4-alcP2r-rTc96RJZtQt4dOg2vpNiYfLDZsV5chXQn_me1nqRFoxQknOh1v1tr_IpCa0pVBkh8-lb5WGxhyJfTof-A2u6NZRAPTTYOk3iZCrCZCKPdX-GeAWlwKW-dssi8q0s2w7GMzN5n8RjHAqC3N7lsv1nc-pqv51j78Cy2nappVew56RgdWw=w611-h494-no?authuser=2)

ROSETTA:
We began to use Rosetta to model and create stable interactions between the nitrogenase and superoxide dismutase complexes. Please see the [Engineering Page](https://2020.igem.org/Team%3ACU-Boulder/Engineering) for code snippets and output from PyRosetta.

BROWNDYE:
In the future, we will use Browndye, a simulation software that uses Brownian Dynamics, to simulate the likelihood of a particle hitting the active site of the nitrogenase enzyme from a specified distance. We will use this program to simulate the probability of a radical oxygen molecule being converted by the SOD complex, rather than inhibiting and destroying the active site of nitrogenase.

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:CU-Boulder/Results

# Conclusion

Our research focuses on creating a complex of a superoxide dismutase (SOD) attached to a nitrogenase to create self-fertilizing plants, and thus decrease the use of harmful fertilizers used worldwide. Our work lays the groundwork for the future steps we intend to take in the near future. As we work and build expertise on different programs, such as BrownDye and PyRosetta, we can start to run simulations on various designs for the intended complex. These programs can help us determine many aspects of the compound, such as attaching sites and the best configuration possible. We will also consider the different types of nitrogenase that arise from different bacteria to determine which one would be most appropriate and beneficial. After deciding on all the aspects and details of the compound through extensive simulation and further research, we will proceed to make our design a reality.