This year, we constructed and characterized multiple sets of parts all registered on the iGEM Parts Registry according to the BioBrick standards. Our parts are briefly listed in this page and categorized according to modules and collections they are in. For detailed information, visiting our Parts Registry page is highly recommended.

Rhodioloside Production Module (Fermentation Module)

BBa_K4761000, BBa_K4761001, BBa_K4761002, BBa_K4761003 and BBa_K4761004 are the basic parts we mainly use for rhodioloside production. BBa_K4761200, BBa_K4761201, BBa_K4761202 and BBa_K4761203, assembled from the basic parts above and able to function smartly, are the parts we actually use during constructions of engineered bacteria. Their introduction and functions are listed below.

Basic Parts

Composite Parts

Copy Number Optimization Module

Original plasmids we use (pSA-ARO10-adh6, pET-UGT85A1, pCS-pgm-galu) have low copy numbers and are supposed to have poor performance. To solve this issue, we tried to link the generator units to different plasmid backbones. The plasmids we use include the former ones BBa_K4761100, BBa_K4761101 and BBa_K4761102 assembled by enzyme linking, and new dual expression vectors BBa_K4761103, BBa_K4761104, BBa_K4761105 using Gibson Assembly®. The vectors used in this part are shown below.

Gene Knockout Module

pheA, pykA, pykF, whose expression may compete tyrosine with rhodioloside pathway we constructed, are the desired genes we planned to knock out. We designed multiple sgRNAs to knockout the corresponding genes using CRISPR-Cas9 method, and constructed multiple pTargets using overlapping PCR.

For knockout of pheA, we tried BBa_K4761020 and BBa_K4761021 the two sgRNAs and constructed pTargets BBa_K4761420 and BBa_K4761421.

For knockout of pykA, we tried BBa_K4761022, BBa_K4761023 and BBa_K4761024 the three sgRNAs and constructed pTargets BBa_K4761422, BBa_K4761423 and BBa_K4761424.

For knockout of pykF, we tried BBa_K4761025, BBa_K4761026, BBa_K4761027 and BBa_K4761028 the four sgRNAs and constructed pTargets BBa_K4761425, BBa_K4761426, BBa_K4761427 and BBa_K4761428.

All the pTarget derivatives are built based on the original one BBa_K4761107. CRISPR-Cas9 dual plasmid system is used.

Fusion Expression Module

In BBa_K4761200, the ARO10 and adh6 gene encodes two different independent enzymes. By constructing them into a fusion protein with linkers, it is supposed to enhance the rhodioloside production. In this module, we designed six different linker sequences BBa_K4761030 to BBa_K4761035, and made tyrosol generation units BBa_K4761220 to BBa_K4761225 using fusion PCR. Their profiles are listed below.

Basic Parts

RNA Thermometer Module

We designed multiple RNA thermometers which could be activated when temperature reaches 37℃. At first, we used BBa_K4761040 to BBa_K4761044 and built report units BBa_K4761230 to BBa_K4761234 to install on pET plasmid (BBa_K4761101 ). Luminescence signal could be detected only by microplate readers. Later new thermometers BBa_K4761045 to BBa_K4761049 are designed and report units BBa_K4761235 to BBa_K4761239 are built to install on pET28a (BBa_K4761106 ) to observe luminescence under naked eye. EGFP (BBa_K4761005 ) is selected to be our reporter protein due to its excellent luminescence density.

Basic Parts

Composite Parts

Gene Integration Module

To research about the effect of integrating our rhodioloside production module into the genome of our chassis, ARO10 and UGT85A1 knock-in strain are constructed using CRISPR-Transposon three-plasmid system. We constructed the corresponding transposon units using Gibson Assembly®. The parts we used are listed below.

Basic Parts

Composite Parts

Primer Parts

The following is a brief list of important primers we used. We list and register them for the convenience of repeat experiments by those who may have interest in our project. For usage and detailed sequence, etc., please refer to their Registry pages. Other primers, including most verification primers and those sharing much resemblance with parts mentioned above, are not registered. Design guidance for these primers is written in corresponding Registry pages instead.

To check all the primers and their numberings we use, please see Primer List page.

Copy Number Optimization Module

Gene Knockout Module (Primers for Donor Synthesis)

Gene Intergration Module

RNA Thermometer Module