Overview

On this page, we present in time order how we developed the engineering design cycle of our project. We have made several rounds of improvement to our project, it benefits us a lot.

Cycle1 TMA Degradation Pathway

Design 1:How we choose the TMA degradation pathway?

In order to reduce the amount of TMA in human gut, we considered to degradate TMA which produced by gut microbiota.

To begin with, we have done a lot of preliminary work, like searchting information and obtaining inspiration from other teams. It was from a review[1] that we learnt there are mainly three ways of TMA degradation in human intestine:The archaeal methanogenic pathway、Stepwise dehydrogenation pathway of TMAandThe aerobic oxidation pathway.The Aslma 2019 had done an hard job, choosing the archaeal methanogenic pathway. As archaea has a complex structure, they only expressed the mttBC and mtbA gene clusters, which contributes to metabolize trimethylamine into methane. At the same time, another team had expressed trimethylamine dehydrogenase and formaldehyde dehydrogenase to metabolize trimethylamine into dimethylamine and transform the byproduct formaldehyde into formic acid. We know from a literature that dimethylamine may have an impact on human health after excessive content[2].Considering the safety of our design, we expressed dimethylamine dehydrogenase to degradate dimethylamine into monomethylamine. It must to be noted that we originally wanted to constructe metabolism monomethylamine-amine passway, while we looked up some literatures and found that the cofactor TTQ (Tryptophan Tryptophylquinone) Escherichia coli did not exist. And the gene cluster required for MMADH expression is too long[3], so it's unlikely to be expressed successfully. Therefore, we chose to express trimethylamine dehydrogenase and dimethylamine dehydrogenase only.

Build 1

Trimethylamine dehydrogenase and dimethylamine dehydrogenase are uncommon in Escherichia coli. Through previous design and discussion, we finally selected gene tmd and dmd fromMesorhizobiumand Hyphomicrobium expressing trimethylamine dehydrogenase and dimethylamine dehydrogenase respectively.

Test 1

After the BL21(DE3) protein expression vector was successfully constructed, we considered how to detect the enzyme activity. As can be seen from the references, we firstly selected the spectrophotometer to detect the enzyme activity. We designed some sets of experiments, but unfortunately, under the conditions of induction with different theophylline concentrations, the activity difference of the two enzymes were not significant.

Learn 1

We analyzed that the measurement accuracy of the spectrophotometer is too low. We should choose to use some methods with higher precision for measurement, like HPLC.

Redesign 1

High performance liquid chromatography (HPLC) is a test method with high accuracy for small molecules. There have been studies on the determination of TMA and DMA by HPLC. Based on the actual situation of the project, we can detect the enzyme activities of trimethylamine dehydrogenase and dimethylamine dehydrogenase by the rise or fall of DMA concentration. Therefore, we finally chose to detect the concentration change of DMA concentration by HPLC.

Rebuild 1

We firstly made standard curves for DMA standard solutions to give a reference for subsequent enzyme activity measurement. For trimethylamine, in order to simulate the situation in the patient's body, the concentration of trimethylamine in the patient's gut was selected as the initial value, and then the enzyme activities of trimethylamine dehydrogenase and dimethylamine dehydrogenase were measured respectively. For the trimethylamine dehydrogenase with original sequence and V344C mutant, we measured the DMA concentration of samples at intervals after induction by adding the initial concentration of TMA. For dimethylamine dehydrogenase, assuming that all TMA can be converted to DMA, we added the same initial concentration of DMA after induction for a certain period of time, and measured the reduction of dimethylamine concentration at different times.

Retest 1

In the process of preparing the standard, we selected DMA solution with LB to control the variabilities. After changing the concentration gradient, we found that the retention time was about 7.0, which corresponding to our DMA concentration through HPLC. The result is shown in the following figure.

According to the standard solution, we analyzed the data of pure BL21(DE3) in the control group, tmd-transferred BL21(DE3) and V344C mutant BL21(DE3), finding that trimethylamine dehydrogenase can be expressed and actived normally, with around 50% express efficiency after 9h of cultivating and inducing. In addition, it was also obviously observed that the activity of the tmd mutant was improved compared with the wild-type, which was consistent with the results of our model part.

Relearn 1

In this part, we learned the usage of HPLC and the measure skills of enzyme activity. Although we did not select the appropriate measure method and the concentration gradient made by the standard curve in the experimental design, but we finally got the preliminary experimental results.

However, it is undeniable that our standard solution contains LB medium, and the growth and metabolic substances of bacteria will also cause errors, so we can conduct further experimental discussion in the future.

Cycle 2 Nattokinase Expression

Design 2: How we express the nattokinase successfully?

Through the study of literature, we found that mentioned in the literature a can be produced in e. coli with the activity of natto kinase, rather than the form of inclusion body, natto kinase sequence is composed of three parts, as PelB secretion signal peptide, natto kinase propeptide, natto kinase mature peptide, natto kinase propeptide help natto kinase to fold, form a ripe natto kinase, PelB secretes nattokinase into the periplasmic space, reduces intracellular nattokinase, and prevents nattokinase from aggregating to form inclusion bodies, thereby successfully secreting mature and active nattokinase.

Build 2

Considering the natto kinase not only has the effect of thrombolysis, also with antioxidant, prevent alzheimer's disease, relieve retinal angiogenesis, anti-cancer, inhibiting inflammation, and so on, at the same time, natto kinase as extract protein from food and less side effects, because in 37 ℃ at the same time, its expression quantity is low, so we will natto kinase as a type of expression.

Test 2

During the experiment, we found that the desired mature and active nattokinase could not be obtained at 37℃ according to the IPTG of 0.7mmol/L in the literature. It was mentioned in the literature that nattokinase would form inclusion bodies in Escherichia coli at 37℃, and the mature nattokinase could not be expressed or the expressed protein was not active. But documents mentioned in 0.7 L is the tendency for testing, it found that the best induced concentration, produces the most amount of natto kinase, which means in the literature about the relation of natto kinase output and temperature is in the best concentration of induced mapping, we know that high temperature will affect the efficiency of protein folding, after consulting the related enzyme engineering teacher, We found that the expression of nattokinase could be reduced to make the expressed nattokinase have enough space and time to fold correctly. Then we decided to adjust the induced concentration of Escherichia coli at 37℃ to 0.1mmol/L according to the difference of protein expression of IPTG at different concentrations in the literature. Finally, we obtained nattokinase which can be correctly expressed at 37℃ and has activity.

Learn 2

Since we expressed nattokinase as a constitutive form, we selected a combination of the weaker promoter and RBS to reduce the expression of nattokinase so that it could correctly express active nattokinase at 37 ° C. In addition, through literature review, we can use fermentation medium to help E. coli to express nattokinase. However, due to the time problem, this part of the design has not been tested experimentally.