Difference between revisions of "Part:BBa K4274032"

Revision as of 11:08, 12 October 2022

ptac-RiboJ-B0034-CmR29M2_ClSS_S533A-B0034-ERG20_F96W-B0015

This composite part was used for the pathway IPP ⇄DMAPP → GPP → FPP → alpha-santalene, which ultimately creates alpha santalene, the precursor of santalol. In this part, we encoded the CmR29M2_ClSS_S533A (C. lansium santalene synthase with a hydrophilic DNA fragment) (Part: BBa_K4274001) and ERG20_F96W (Part: BBa_K4274002), an enzyme that catalyzes the formation of FPP (farnesyl diphosphate). Besides, the IPTG inducible promoter ptac-RiboJ (Part: BBa_K3552015) and the ribosome binding site B0034 (Part: BBa_B0034) were utilized for the expression of CmR29M2_ClSS_S533A and ERG20_F96W.
Then, the composite part ends with the terminator B0015 (Part: BBa_B0015). Our parts collection aims to optimize the production of alpha-santalene. There are two parts in this collection aiming to optimize the activity of FPP (farnesyl diphosphate), respectively ERG20_F96W (Part: BBa_K4274002) and ptac-RiboJ-B0034-ClSS_S533A-B0034-ERG20-B0015 (Part: BBa_K4274030). Then, we also composed parts that secretes ClSS and ERG20, comprising this composite part (BBa_K4274032) and CmR29M2_ClSS_S533A (PART: BBa_K4274001). Finally, we have two parts exploring the effects of adding a flexible linker and the production of alpha-santalene, consisting of ClSS_S533A-FL-ERG20_F96W (Part: BBa_K4274033) and (Part: BBa_K4274003). By organizing this parts collection, we hope to hope the future teams by establishing our results and explorations on alpha-santalene production, providing some inspirations for in-depth investigation and utilization.

Usage and Biology

CmR29M2_ClSS_S533A is a gene sequence that expresses C. lansium santalene synthase, an enzyme that catalyzes the transformation from FPP (farnesyl diphosphate) to alpha-santalene. Two modifications have been made that made this part. To begin with, there was an artificial mutation that mutated the 533rd amino acid of ClSS from serine to alanine. Then, we have also attached a DNA fragment derived from Synechocystis, performing the role of a solubility enhancer (Nico Betterle et al., 2018), hoping to increase the expression of ClSS by improving its solubility. It was concluded by research that adding CmR29M2 can lead to 131% production of alpha-santalene comparing to the ones with no tag (Jia Z et al, 2022). However, after conducting a series of experiments and tests, our results demonstrate that adding a tag to enhance solubility does not increase the production of alpha-santalene. On the contrary, it decreases the production to only 1/3 of the production without a tag strain. Therefore, the addition of CmR29M2 is not effective at optimizing the production of alpha-santalene. In addition, we have also expressed ERG20_F96W in this composite part. ERG20_F96W is an enzyme that catalyzes the formation of FPP (farnesyl diphosphate). Its 96th amino acid was artificially mutated from phenylalanine to tryptophan.

Source

ClSS_S533A (Part: BBa_K4274000) is from Clausena lansium, ERG20_F96W (Part: BBa_K849001) is from S. cerevisiae, and CmR29 is from Synechocystis.

Sequence and features

Sequence and Features