Updated on 2022-10-28: We won Best New Basic Part, and were nominated for Best New Composite Part this year.
In our project, over sixty parts were planned and 52 were obtained. Among them, there are 10 basic parts and 42 composite parts. We have sequencing verified all parts and tested most of the parts.
Basic parts
Among the 10 basic parts designed this year, BBa_K4162005 (Hammerhead Ribozyme) is our favorite one.
We construct a ribozyme-assisted polycistronic co-expression BioBrick (pRAP) by inserting ribozyme sequences between CDSs in a polycistron. Using pRAP, the RNA sequences of hammerhead ribozyme conduct self-cleaving, and the polycistronic mRNA transcript is thus co-transcriptionally converted into individual mono-cistrons in vivo. Self-interaction of the polycistron can be avoid and each cistron can initiate translation with comparable efficiency. Besides, we can precisely manage this co-expression system by adjusting the RBS strength of individual mono-cistrons. In our project, we used ribozyme to build our crtEBIY BioBrick.
For more information, please click BBa_K4162005: Favorite Basic Part.
Composite parts
Our favorite composite parts are BBa_K4162117 and BBa_K4162117.
BBa_K4162117: This BioBrick was created through overlapping PCR of BBa_K4162020 (ribozyme+J6_RBS+crtY), BBa_K4162010 (ribozyme+T7_RBS+crtE), BBa_K4162013 (ribozyme+T7_RBS+crtB) and BBa_K4162016 (ribozyme+T7_RBS+crtI). In this part, the RBS of crtEBI has equal intensity while the RBS of crtY is significantly weaker than the others. Because crtY catalyzes the last step of the carotenoid reaction chain, we guess the concentration of substrate catalyzed by this enzyme is significantly lower than for the first three steps of the reaction. To avoid the problem of flux imbalance in biosynthesis as well as to reduce unnecessary metabolic stress on cells, we intentionally weakened the RBS intensity of crtY.
Figure 1. BBa_K4162117
BBa_K4162118: This BioBrick was created through overlapping PCR of BBa_K4162020 (ribozyme+J6_RBS+crtY), BBa_K4162010 (ribozyme+T7_RBS+crtE), BBa_K4162013 (ribozyme+T7_RBS+crtB) and BBa_K4162016 (ribozyme+T7_RBS+crtI). In this part, the RBS of crtBIY has equal intensity while the RBS of crtE is significantly stronger than the others. Since crtE catalyzes the first step of the carotenoid reaction chain, increase the concentration of product catalyzed by this enzyme is beneficial for the remaining three steps. To avoid more serious flux imbalance problems, we boosted the RBS intensity of crtE only in this BioBrick and explored whether the carotenoid production of the strain could be significantly enhanced.
Figure 2. BBa_K4162118
Figures 3 and 4 show that transfected E. coli successfully expressed the target enzyme and yielded β-carotene. In Figure 3, it can be seen that module YEBI corresponds to a darker orange color of the post-centrifugation precipitation compared to module YBEI (BBa_K4162119), characterizing the superior carotenoid yielding ability of module YEBI. As shown in Figure 4, different clones of E. coli transformed with the same plasmid were cultured using 96-well plates. After shaking culture overnight, the plate was spin at 4500 rpm for 30 minutes and bacteria was pelleted to the bottom of the wells. There is no clonal variation, showing stable expression of the individual mono-cistrons separated by ribozyme sequences (i.e., no trucated expression nor null function protein of downstream genes due to being placed under one promoter).
Figure 3. The centrifuge tubes containing module crtYEBI (first from the left) and module crtYBEI BBa_K4162119 (second from the left) contain visible yellow bacterial pellet.
Figure 4. 96-well plate of module crtYEBI. Except for the blank control well marked in black, all clones growing different wells had similar β-carotene content in the bacterial pellet.
For more information, please click BBa K4162117: Favorite Composite Part and BBa K4162118: Favorite Composite Part.
Support from PartHub
In all of our Design-Build-Test-Learn (DBTL) pipelines (see engineering), we use PartHub for gene and similar sequence retrieval.
PartHub is a powerful search tool from us. It is proved to be useful for our experimental work. For more information, please visit our software page.
Below list all the parts we built for our project. Please click the name to see deails on the Registry.
| Part Name | Description | Part Type | Designer(s) | Length | |
|---|---|---|---|---|---|
| BBa_K4162001 | StayGold = (n2)oxStayGold(c4)v2.0 | Coding | Weiwen Chen | 711 bp | |
| BBa_K4162002 | linker between BCMO and StayGold, hydrophobic | Coding | Weiwen Chen | 54 bp | |
| BBa_K4162003 | ybbO coding for retinol dehydrogenase, codon optimized for E. coli | Coding | Yujian Song | 810 bp | |
| BBa_K4162004 | Gene coding for BCMO from uncultured marine bacteria | Coding | Weiwen Chen | 828 bp | |
| U | BBa_K4162005 | Hammerhead ribozyme | RNA | Weiwen Chen | 57 bp |
| BBa_K4162006 | T7_RBS | RBS | Weiwen Chen | 17 bp | |
| BBa_K4162007 | MCP | Coding | Weiwen Chen | 336 bp | |
| BBa_K4162008 | tdMCP | Coding | Weiwen Chen | 690 bp | |
| BBa_K4162009 | ribozyme + B0_RBS + crtE | DNA | Weiwen Chen | 991 bp | |
| BBa_K4162010 | ribozyme + T7_RBS + crtE | DNA | Weiwen Chen | 993 bp | |
| BBa_K4162011 | ribozyme + J6_RBS + crtE | DNA | Weiwen Chen | 981 bp | |
| BBa_K4162012 | ribozyme + B0_RBS + crtB | DNA | Weiwen Chen | 1012 bp | |
| BBa_K4162013 | ribozyme + T7_RBS + crtB | DNA | Weiwen Chen | 1014 bp | |
| BBa_K4162014 | ribozyme + J6_RBS + crtB | DNA | Weiwen Chen | 1002 bp | |
| BBa_K4162015 | ribozyme + B0_RBS + crtI | DNA | Weiwen Chen | 1561 bp | |
| BBa_K4162016 | ribozyme + T7_RBS + crtI | DNA | Weiwen Chen | 1563 bp | |
| BBa_K4162017 | ribozyme + J6_RBS + crtI | DNA | Weiwen Chen | 1551 bp | |
| BBa_K4162018 | ribozyme + B0_RBS + crtY | DNA | Weiwen Chen | 1231 bp | |
| BBa_K4162019 | ribozyme + T7_RBS + crtY | DNA | Weiwen Chen | 1233 bp | |
| BBa_K4162020 | ribozyme + J6_RBS + crtY | DNA | Weiwen Chen | 1221 bp | |
| BBa_K4162021 | ribozyme+RBS+CDS module: crtIYEB | DNA | Weiwen Chen | 4896 bp | |
| BBa_K4162022 | ribozyme + T7_RBS + BCMO | DNA | Weiwen Chen | 909 bp | |
| BBa_K4162023 | ribozyme + T7_RBS + ybbO | DNA | Weiwen Chen | 891 bp | |
| BBa_K4162024 | ribozyme + T7_RBS + BCMO + ribozyme + T7_RBS + ybbO | DNA | Weiwen Chen | 1800 bp | |
| BBa_K4162025 | ribozyme + T7_RBS + ybbO + ribozyme + T7_RBS + BCMO | DNA | Weiwen Chen | 1800 bp | |
| BBa_K4162026 | ribozyme + T7_RBS + BCMO + ribozyme + T7_RBS + ybbO-tdMCP | DNA | Weiwen Chen | 2508 bp | |
| BBa_K4162027 | ribozyme + T7_RBS + ybbO-tdMCP | DNA | Weiwen Chen | 1599 bp | |
| BBa_K4162028 | ribozyme + T7_RBS + ybbO-tdMCP-EGFP | DNA | Weiwen Chen | 2340 bp | |
| BBa_K4162029 | ribozyme + T7_RBS + BCMO + ribozyme + T7_RBS + ybbO-tdMCP-EGFP | DNA | Weiwen Chen | 3249 bp | |
| BBa_K4162031 | ribozyme + T7_RBS + BCMO-StayGold | DNA | Weiwen Chen | 1674 bp | |
| BBa_K4162032 | ribozyme + T7_RBS + BCMO-StayGold + ribozyme + T7_RBS + ybbO | DNA | Weiwen Chen | 2565 bp | |
| BBa_K4162033 | linker between gene ybbO and tdMCP | DNA | Weiwen Chen | 18 bp | |
| BBa_K4162034 | linker between tdMCP and EGFP | DNA | Weiwen Chen | 18 bp | |
| BBa_K4162101 | ribozyme+RBS+CDS module: crtIY | Coding | Weiwen Chen | 2796 bp | |
| BBa_K4162102 | ribozyme+RBS+CDS module: crtEI | Coding | Weiwen Chen | 2556 bp | |
| BBa_K4162103 | ribozyme+RBS+CDS module: crtBE | Coding | Weiwen Chen | 2007 bp | |
| BBa_K4162104 | ribozyme+RBS+CDS module: crtEB | Coding | Weiwen Chen | 2007 bp | |
| BBa_K4162105 | ribozyme+RBS+CDS module: crtYB | Coding | Weiwen Chen | 2247 bp | |
| BBa_K4162106 | ribozyme+RBS+CDS module: crtIB | Coding | Weiwen Chen | 2577 bp | |
| BBa_K4162107 | ribozyme+RBS+CDS module: crtEY | Coding | Weiwen Chen | 2226 bp | |
| BBa_K4162108 | ribozyme+RBS+CDS module: crtEBI | Coding | Weiwen Chen | 3570 bp | |
| BBa_K4162109 | ribozyme+RBS+CDS module: crtIEB | Coding | Weiwen Chen | 3570 bp | |
| BBa_K4162110 | ribozyme+RBS+CDS module: crtBEY | Coding | Weiwen Chen | 3240 bp | |
| BBa_K4162111 | ribozyme+RBS+CDS module: crtBEI | Coding | Weiwen Chen | 3570 bp | |
| BBa_K4162112 | ribozyme+RBS+CDS module: crtIYB | Coding | Weiwen Chen | 3810 bp | |
| BBa_K4162113 | ribozyme+RBS+CDS module: crtEBY | Coding | Weiwen Chen | 3240 bp | |
| BBa_K4162114 | ribozyme+RBS+CDS module: crtYEB | Coding | Weiwen Chen | 3240 bp | |
| BBa_K4162115 | ribozyme+RBS+CDS module: crtYBE | Coding | Weiwen Chen | 3240 bp | |
| BBa_K4162116 | ribozyme+RBS+CDS module: crtEIY | Coding | Weiwen Chen | 3789 bp | |
| U | BBa_K4162117 | ribozyme+RBS+CDS module: crtYEBI | DNA | Weiwen Chen | 4836 bp |
| U | BBa_K4162118 | ribozyme+RBS+CDS module: crtEBIY | DNA | Weiwen Chen | 4848 bp |
| BBa_K4162119 | ribozyme+RBS+CDS module: crtYBEI | DNA | Weiwen Chen | 4836 bp |