This year ZJU-China innovatively stepped out of the therapeutic or diagnostic track that we have often done in previous years. Instead we explored new applications of synthetic biology in heritage conservation. But this made our project topic quite different from the topics our previous partners chose, so establishing a proper Partnerhip became a major problem for us this year. Fortunately, we were approached by CAU_China, who also dedicated to heritage conservation. We held our first online Meetup in early May to introduce each other to the team's prototype project design.
ZJU-China's project focused on restoring tiny cracks of stone artifacts, while CAU_China focused on weathering conservation of stone artifacts. And both teams intended to use biomineralization techniques in their projects. More amazingly, the inspiration of CAU_China's project , Lingyin Temple, is located in Hangzhou, less than 5km away from ZJU campus.
With similar background, CAU_China hit the nail on the head to us that, the questions that whether the growth state of the engineered bacteria is susceptible to environmental changes and whether the microorganisms can cause secondary damage to the artifacts, should be taken into account when building our project. This inspired us to develope a hardware for monitoring the restoration process.
Fig 1 Our first online meetup with CAU_China
When the summer holidays started, the experiments had been going on. As most of the team members were new to molecular cloning experiments, both ZJU-China and CAU_China encountered some problems. In this case, the two teams had another Meetup to share our experimental progress and try to solve the difficulties we met during laboratory works.
CAU_China mentioned that when they tried to insert more than one fragments into the plasmid at the same time, they often ligated three exogenous fragments with Gibsson first, but the bands of the ligated products could not be observed by agarose gel electrophoresis after ligation. As they said, this problem had plagued them for a long time, almost causing the progress of the experiment to stall. In response, ZJU-China suggested that the plasmid backbone could be ligated with three exogenous fragments in the same system by Gibsson, and the products could be used directly to transform E.coli after ligation. Our suggestion was validated in subsequent experiments, and all subsequent Gibson assemblies performed by CAU_China were successful, which allowed CAU_China to save a lot of time from ligating fragments two by two by Overlap PCR.
Fig 2 Inserting more than one exogenous fragments to the plasmid backbone using Gibson
At the same time, ZJU-China suggested that we had encountered some problems in testing the biosafety module. We planned to introduce the MazE/F module downstream of the sucrose-inducible promoter PsacB, but found that the sucrose-inducible activity of PsacB was not sufficient to test the suicide function of MazE/F. Thankfully, CAU_China was willing to help us to measure the MazE/F module, which also provided them with a biosafety solution. ZJU-China, on the other hand, worked on improving the induction activity of PsacB promoter. CAU_China has provided us with the results of the MazE/F module characterization, easing our experimental workload.
Fig 3 CAU_China is characterizing the MazE/F module
CAU_China introduced MazE/F under control of Threonine-sensitive promoter. The result we expect is that the engineered bacterial will survive under threonine induction and die with the absence of threonine. But the experimental result showed that whether threonine existed or not, the engineered bacteria always had a mortality of nearly 86%. There were also several times that the colony they picked from the selecting plate would not grow in liquid medium.
We ZJU-China had encountered a similar problem in the early stages of experiments that the transformed bacteria on selecting plate cannot grow in liquid medium. When discussing with CAU_China, we started to think that this situation was caused by leaked expression. Also CAU_China presumed the design of MazE/F under control of one single promoter was not reasonable. But in the middle of this discussion, Yan Zhijian from ZJU-China had an insight that this phenomenon might be caused by the variation of promoter activity in different growth stages. He said, "As Bacillus subtilis has 18 σ factors, different promoters have different activities in different growth stages. The engineered bacteria might die because of the high promoter activity just after transformation." CAU_China strongly agrees with this idea. In addition, the variation in promoter activity can also lead to the imbalance between concentrations of toxin and antitoxin, which could explain why the bacteria containing this suicide pathway are extremely unstable.
Fig 4 CAU_China is measuring the MazE/F module
During the exchange between the iGEM teams, we found that CAU_China is similar to us in this aspect of discrete process modelling. Both of us are using cellular automation. We discussed the characteristics of cellular automation and the rules for their use, which helped both our teams to make more rational use of this model. In biochemical reactions, the Michaelis-Menten equation requires many enzyme-related coefficients, and CAU_China gave a website where these coefficients are more easily available, which greatly improved the efficiency of our modelling. Also, in the analysis of the results of the model, we discuss the method of testing the sensitivity, which provides a good help to the validity of our model.
As CAU_China was inspired by the millennium-old white pagoda of Lingyin Temple in Hangzhou, which is located less than 5km away from ZJU campus, ZJU-China made efforts to learn more about the recent research of the natural calcium oxalate protective film on this pagoda. We were surprised to find that, the first scholars who discovered and tried to synthesis this protective film were the team led by professor Zhang bingjian from the School of Art and Archaeology of ZJU. We immediately emailed Professor Zhang, and were invited to visit their laboratory.
Fig 5 School of Art and Archeology, ZJU
We learned that Professor Zhang's team had taken samples from the white pagoda of Lingyin Temple and confirmed that chemical composition of this transparent protective film was calcium oxalate monohydrate. Professor Zhang's team also tried to synthesis this calcium oxalate film artificially, but found that the synthetic film did not adhere tightly to the surface of the stone artifacts, failing to provide adequate protection. They then tried to replace calcium oxalate with calcium phosphate, or replace its Ca2+ with Mg2+ , but all approaches did not show satisfying results. ZJU-China shared what we have learnt from Professor Zhang to CAU_China in the hope that it would help them improve their project design.
Fig 6 ZJU-China with Professor Zhang’s team
At the beginning of the project, ZJU-China was still unsure whether to focus on restore large cracks or tiny cracks for our project, so CAU_China used their existing Human Practice results to help us gather images and analysis of the problems faced by stone artifacts around the world. We discussed and concluded that there are already a number of restoration methods for larger cracks, so our project is better suited to focus on tiny cracks to enhance our creativity and competitiveness. These efforts enriched the context of our project.
Fig 7 Tiny cracks of stone artifacts
As mentioned earlier, the inspiration of CAU-China's project was the white pagoda at Lingyin Temple in Hangzhou. This summer, some team members of CAU_China had the opportunity to travel to Hangzhou to meet with ZJU-China and visit Lingyin Temple together, investigating the preservation of stone artifacts in the wild.
Located in Hangzhou, Zhejiang Province, Lingyin Temple was built in 326 A.D. and is now a key cultural relic protection unit in China, preserving over 200 limestone artifacts. The twin pagodas to the left and right of Lingyin Temple are the White Pagodas, which we have heard so much about and which have been 'imperishable for a thousand years'. During this offline visit, we found that the pagodas are partly covered with a natural calcium oxalate protective film, but the uncovered surface in the open air still shows signs of weathering and erosion. The inscriptions were also blurred.
Fig 8 ZJU-China and CAU_China visiting white pagoda at Lingyin Temple in Hangzhou.
We discussed that the damage to the stone artifacts in the open air of the Lingyin Temple in Hangzhou was probably due to two factors, (i) the vegetation in Lingyin Temple is luxuriant and biological communities such as moss and lichen can usually be found around the artifacts, which may have enriched the surface of the artifacts with acidic substances causing erosion. (ii) The high level of low pH rainfall in Hangzhou may have caused weathering of the stone artifacts due to the long-term washout of acid rain.
We observed that cracks and damage on stone pagodas had been repaired by teams using glutinous rice mortar. Glutinous rice mortar is a traditional Chinese building technique that uses a very dilute glutinous rice paste mixed in with restoration filler, which is not only strong and antibacterial, but also has some self-healing ability. Even if the cracks develops again, the calcium ions in the filler can be dissolved and re-cured, ensuring the longevity of the restoration. However, its appearance is not fully compatible with stone artifacts and can cause some cosmetic impact. After discussion, ZJU-China and CAU_China arrived at several principles for the restoration and conservation of cultural objects.
(i) The materials chosen should have good compatibility with the substrate materials of the artifact, preferably with ease of removal, leaving enough room for future technological advances to allow future conservation with better materials or methods.
(ii) Obey the 'principle of minimal intervention', where the extent of damage does not require restoration, we shouldn’t interfere with manual measures, thus to prevent secondary damage to cultural objects caused by inappropriate measures.
Fig 9 ZJU-China and CAU_China investigating the preservation of stone artifacts in the wild.
iGEM is an opportunity to promote synthetic biology to the public, so CAU_China and ZJU-China, in collaboration with Beijing Youth Exchange, conducted an online lecture on synbio for young university students in Beijing. Starting from a map of the world's stone artifacts, we introduced the classical operational techniques of synthetic biology. More than 200 participants were involved. After this lecture, we received positive feedback from the participants.
Fig 10 ZJU-China and CAU_China giving online lecture
As it is the first time for both CAU-China and ZJU-China team members to participate in the iGEM, there is a lot of uncertainty about the competition process, deadlines, and details of the requirements. Therefore, we discussd with CAU_China whenever there were unclear issues. For example, when submitting the safety form, we discussd the safety of the chassis organisms we use, and when choosing tracks, we analysed which track fits our projects the most. We also attended meetups for Chinese iGEMers together to absorb relevant experience when listening to other teams' presentation.
From the brainstorming to the experimental validation as well as human practice, we ZJU-China has had close communication and cooperation with CAU_China. Both teams have provided each other with indispensable assistance. We are sincerely grateful to CAU-China for being a part of our journey and achieving the standard of the golden medal with us.
