Team:HZAU-China/Labnotes

Parts connection and standardization:

Author: Zheng Yihui

Members: Zheng Yihui, Zhang Yajing, Liu Zhixiang, Luo Xudong, Guo Xiaoxue, Wang Lu, Li Siqi.

Introduction:

We have eight gene circuits to construct (as shown in the figure.1), and after finishing these circuits, we will integrate some of them into one plasmid to form two repressilators and two detection devices (as shown in the figure.2a and figure.2b).

May

Content: we found the parts we needed in the Plate Kits and did a lot of transformations to get the plasmid for subsequent experiments.

Parts list: BBa_J06602 BBa_E0420 BBa_K886000 BBa_R1051 BBa_P0412 BBa_R0040 BBa_P0451 BBa_R0011 BBa_R0051 BBa_I714075 BBa_J61047 BBa_R0063 BBa_K516022 BBa_K805016 BBa_J37032 BBa_K081019 BBa_K773003 BBa_I13602 BBa_R0061 BBa_R0062 BBa_R0063 BBa_C0060 BBa_B0015 BBa_I718016 BBa_I0462 BBa_P0440 BBa_E0022 BBa_J01010

June

Content: eight gene circuits’ construction

Construction details:

1.Construction of gene circuit one: we used standard method of biobrick connection to put the coding sequence of cI protein behind PLlacI promoter. But we failed to construct it because of some unknown reasons. The sequence results showed that there is either a pretty long and unknown segment which insert into the coding sequence of cI protein or deficiency of the sequence of PLlacI promoter(as shown in the figure.3). So, we had an assumption that there were little LacI proteins in DH-5α, and it was not much enough to repress PLlacI promoter, resulting in the overexpression of cI protein which may restrain the growth of host.

2.Construction of gene circuit three: Firstly, we used standard method of biobrick connection to put the coding sequence of LacI protein behind lox66 because the length of lox66 was 34bp and it was too small to purify from the enzyme-digested product and then put it in front of LacI protein. Due to the same reason, we put the product of previous step behind PLtet01 promoter to finish this gene circuit. Compared to construction of other gene circuits, it seemed that this gene circuit was the simplest job we’ve made since that we only tried once and took almost eight days to finish this work.

3.Construction of gene circuit two: we constructed this gene circuit by following the method we’ve mentioned above. The only difference from the construction of gene circuit three is that we put the coding sequence of tetR protein behind lox71. By the way, lox66 and lox71 are two recombination sites which are recognized specifically by Cre protein. To our surprise, when we did double-digest to this circuit for verification, we found that the length of product of enzyme-digested was not right and there was an extra stripe that we didn’t know what it was. Results are shown in the figure.4. According to the results, we had another assumption to explain the phenomenon: there were some recombination systems in DH-5α, resulting in homologous recombination of two strains which had synclastic lox71 site. Definitely, We’ve done a lot of work to verify our assumption which will be explained in the part of characterization.

Our project includes three systems---input system, processing system and output system, which are constituted by eight gene circuits, and each circuit is composed of at least three parts(as shown in the design overview of our project). We take almost four months to construct all of them by the standard method of biobrick connection and test them whether they work as expected or not by detecting fluorescence intensity, and definitely, it is totally a long and tough road. During the process, we conduct all kinds of experiments like double digestion, ligation, transformation and even some chemical experiment to put one gene in front of or behind another gene and synthetize the material we need. Besides, we make our greatest efforts working on testing our parts and analyzing the data we get. Unexpectedly, we find a phenomena related to the unexpected recombination of lox71 in DH5α and of course, we have done some testing to verify our assumption. Anyway, the road to success is laborious, but we still stick to our goal and finally make it by ourselves. The following is the methods of our construction and the record of our experiments (recorded by different writers).