Team:NDNF China/Notebook

Week 1 (6.24-6.30)

The chromoprotein circuit induced by arabinose, the L-dopa expression circuit induced by caffeine and the primers needed for the construction of gene circuit were preliminarily designed, and the primers needed for the construction of mercury ion detection circuit were sent to the company for synthesis.

We also purchased reagents needed for our experiments.

Week 2 (7.1-7.7)

Part one

PCR amplification plasmid backbone and corresponding parts

PCR amplification for RGP plasmid, PSC101 plasmid and pET28a plasmid backbone. However RGP plasmid and PSC101 plasmid were not successfully amplified.

PCR amplification of the rest of the gene circuits: Four chromoprotein genes (gfasPurple, fwYellow, amiICP, spisPink), green and red fluorescent protein genes (sfGFP, mScarlet), mercury ion detection gene elements (chuA,HrtR,pHrt), Caffeine induced Circuit Element (Pbad-PROMOTER, IBMC378-PARTB, HpaB, HpaC)

Part two

First attempt to construct hydrogel core

First attempt to construct hydrogel core: the alginate solution containing bacterial strains was first dripped into the CaCl2 solution to prepare the core. However we found that the core prepared by this method was small, and a better way to prepare the core was needed.

Week 3 (7.8-7.14)

Part one

The chromoprotein expression strain induced by arabinose was successfully constructed

Four chromoprotein circuits induced by arabinose were constructed by Gibson, and sequencing proved that all the circuits were successfully construct. Then the plasmids were preserved in DH5a and transferred into top10 strains to verify the normal functioning of gene circuits. Also, psc101 and RGP plasmid backbone were amplified again.

Part two

Successful construction of a proper sized hydrogel core

We prepared hydrogel cores of appropriate size by dripping alginate solution mixed with bacterial liquid onto parafilm.

However, in the process of preparing the shell, we found that directly using Acr-Bis solution to dissolve alginate could easily cause solidification in the process of dissolution, so it was necessary to change the dissolution method.

Week 4 (7.15-7.21)

Part one

Barcode design complete

Gibson assembly was used in building the mercury ion sensing plasmid and caffeine detection plasmid system that initially used red fluorescent protein (RFP) as an output reporter gene to assist us in identifying whether construction was correct. The construction was later validated by sequencing. Then the plasmids were kept in DH5a strains.

The construction of the caffeine detection plasmid failed. Showed by the sequencing results, some components were not connected to the plasmid and needed to be rebuilt.

Design barcode and send it to company for synthesis.

Part two

The preparation process of shell material was optimized

Through experiments, we finally chose to prepare Hidro shell by fully mixing 10% alginate with ACR-BIS solution in a certain proportion, so that the gel would not solidify in the process of dissolution.

In the process of soaking MES crosslinking system, we found that the shell was loose and easily fell off after soaking for 3h, so it could not form a flexible shell. Therefore, a better method was needed.

Week 5 (7.22-7.28)

Part one

Successfully constructing Hg detection plasmid

Gibson assembly was used to construct the caffeine detection plasmid, which was verified by plasmid sequencing, and then stored in DH5a strains.

The plasmid containing mercury ion detection circuit was transformed into top10 strains for functional test.

PCR amplified the Ptarget plasmid skeleton, barcode, arabinose inducer promoter and toxic protein Doc gene sequences for the future plasmid construction.

Part two

The ingredient formula of Hidro was further optimized

After consulting a large number of literatures and communicating with the author of the original lecture, we added calcium ions to MES crosslinker, and the experimental results show that adding calcium ions can help Hideo to form a tough and not easily fall off shell.

Week 6 (7.29-8.4)

Part one

First attempt to construct Gene editing plasmid

The Ptarget plasmid which was responsible for later incorporation of the barcode, arabinose induced toxic protein expression system into the genome was constructed by Gibson. However, through sequencing results, it was found that the Ptarget plasmid we used was a high copy plasmid, so the DOC protein gene sequence could not be added into the Ptarget plasmid. Therefore, we considered replacing the medium copy plasmid, and at the same time replacing the toxic protein gene.

Part two

Hidro that could achieve biocontainment was constructed

We tested the escape prevention function of our prepared Hidro. Results proved that the hidro still had the ability to prevent the leakage of internal microorganisms after 72 hours incubation in LB medium. However, at the same time, we found that some internal microorganisms in Hidro were unable to survive. Therefore, we want to improve the survival rate of internal microorganisms by reducing the time and measurement of microbial contact with toxic substances in the preparation process.

Week 7 (8.5-6.11)

Part one

The functional verification of mercury ion detection strain was successful

We reselected the toxin protein gene, constructed the Ptarget plasmid, and edited the top10 strains. The sequencing results showed that we successfully edited the genomes of top10 strains.

We tested the function of the previously constructed strain capable of sensing mercury ions in the environment and expressing fluorescent proteins, and the result shows it could work normally in the liquid medium environment.

Part two

Once again optimize the ingredient formula

We improved survival rate of enclosed microbes by making adjustments to shell component percentages, including lowering toxic reagent dosage of acrylamide, increasing dosage of TEMED which promotes crosslinking, reduce the time soaking in toxic MES crosslinking agent, etc. After that, we compared the new formula with the old one, and we found out that the survival ratio of microorganisms in the revised formula was significantly improved while the microbial escape rate was still considerably low.

Week 8 (8.12-8.18)

Part one

The successful functioning of toxic protein was detected, and the caffeine-induced fluorescence producing strain was constructed successfully.

We sent the edited strains to the lab of GBSZ team, where they used Cas12 technology to test the barcode that we edited into the genome.

We tested the function of the toxic protein: Kid production induced by arabinose, and found that the system could function well, resulting in the death of bacteria.

Also, We transformed the caffeine detection plasmid and the caffeine response plasmid with red fluorescent protein as reporter gene into top10 strains, and tested its' function in LB culture. The experimental results proved that the circuit could work successfully as we predicted.

Part two

The arabinose induced color protein circuit and mercury ion detection circuit were proved to be capable of successfully working in Hidro.

We enclosed strains of arabinose induced chromoprotein expressive circuits and mercury ion detection circuit into Hidro for detection, and the results showed that these two strains could successfully function in Hidro.

Week 9 (8.19-8.25)

Part one

Barcode detection success

Our barcode was successfully detected.

We replaced the reporter gene on the caffeine responsive plasmid with L-dopa expression gene, and verified the successful construction of the plasmid by sequencing.

Part two

Strains that sense caffeine molecules and express red fluorescent proteins were proved to normally work in HIDRO.

We enclosed the strains capable of sensing caffeine molecules and expressing red fluorescent protein into Hidro for functional testing, and the results showed that the strain could work normally and expressed fluorescent in Hidro.

Week 10 (8.26-9.1)

Part one

The caffeine-induced L-dopa expressive strain was successfully constructed

We tested the function of the caffeine-sensitive strain expressing L-dopa in LB medium, and the results proved that the strain could function successfully.

Part two

Various kinds of stress resistance of HIDRO were tested

We tested the protective effect of Hidro on internal microorganisms in a variety range of adverse environments, and the result founds that within the range of acidic pH=1.5, alkaline pH=10, at the temperature of 90℃ and drought for 72h, Hidro could provide protection to internal microorganisms in comparson than when directly exposed in liquid medium.

Week11 (9.2-9.8)

The caffeine-induced dopa production circuit and the food spoilage detection circuit were transferred into Hidro for functional verification.

We enclosed the strain that could sense caffeine molecules and express L-dopa into Hidro for functional testing, and the results showed that the strain could work normally in Hidro and secrete L-dopa to external environments.