diff --git a/content/13.hardware.md b/content/13.hardware.md index dc3ff291581c264a958fc06249deabc859f8c147..22ee0ad64b0710921f222cdfebef86d027450d6d 100644 --- a/content/13.hardware.md +++ b/content/13.hardware.md @@ -1,5 +1,6 @@ <center> + # **Hardware** </center> @@ -10,26 +11,34 @@ ## **Introduction** +<br/> + After deciding to design an AND-gate for sensing hypoxia and high lactic acid, we consider how to detect the expression intensity in different oxygen environments and various lactic acid concentrations. A relatively established measurement method is ELISA, but Multi-Mode Microplate Reader (Biotek) lacks control over the concentration of lactic acid and oxygen. In our actual experiment, we culture E. coli in a stable environment for a specific duration before transferring it to Biotek for measurement. The fluorescence intensity data obtained from Biotek also require the exclusion of bacterial culture concentration's influence. In the experiment, the fluorescence intensity is divided by the OD value. We obtain differentiated results by Biotek, but Biotek can only perform fluorescence intensity measurements at a macroscopic level, with relatively lower accuracy. Additionally, the plasmids we introduced into E. coli are of low copy number, resulting in inherently low fluorescence intensity. This increases the measurement error in our results. A more accurate measurement method can enhance the reliability of our data. Our ultimate plan is to employ microfluidic chip technology to acquire single-cell precise fluorescence intensity data at specific oxygen and lactate concentrations. - +<br/> ## **Design of the microfluidic chip** +<br/> + Microfluidic chip need to meet the following functions: +<br/> + 1, There is an oxygen concentration gradient in the chip environment 2, The chip can bind the bacteria to grow in a small area 3, the chip simultaneously measures a variety of E.coli, and the E.coli do not interfere with each other +<br/> + Based on the above three requirements, we make changes on the basis of the previous chip and design a chip that forms six oxygen concentrations and measure four types of E.coli simultaneously (see Figure 1). <br/> -<center><img src="w1.png" alt="Figure 1" style="width: 70%;" /></center> +<center><img src="r2/chipschematic2.jpg" alt="Figure 1" style="width: 70%;" /></center> <font size=3 color=grey> @@ -52,18 +61,19 @@ We design the chamber to be very low, only 1.3 microns, and the chamber outlet i The first step of loading is to plug the culture inlet. The bacteria solution flows in from the bacteria inlet and out through the bacteria outlet. At this stage, a large number of E.coli will be distributed in the flow channel. The second step of loading is to plug the bacteria inlet and open the culture inlet. The culture solution will pass through the trap due to the pressure difference, thereby flushing the E. coli into the trap. This method not only allows real-time updating of the culture fluid in the trap but also enables trapping of the E.coli. - +<br/> ## **Manufacture process of the microfluidic chip** -We first use laser printing to create a mask consisting of five layers of chips (see Figure 3a). Next, we apply a certain height of photoresist and expose it to develop a mold consisting of two layers of chips (see Figure 3b and Figure 3c). PDMS is then spread on the mold to obtain the upper and lower chips, which are subsequently spliced together (see Figure 3d). The chip is then baked overnight in an oven at 70℃. The following day, the chip is punched (see Figure 3e) and combined with the slide (see Figure 3f). Finally, the chip is baked for over 3 hours. - <br/> +We first use laser printing to create a mask consisting of five layers of chips (see Figure 3a). Next, we apply a certain height of photoresist and expose it to develop a mold consisting of two layers of chips (see Figure 3b and Figure 3c). PDMS is then spread on the mold to obtain the upper and lower chips, which are subsequently spliced together (see Figure 3d). The chip is then baked overnight in an oven at 70℃. The following day, the chip is punched (see Figure 3e) and combined with the slide (see Figure 3f). Finally, the chip is baked for over 3 hours. + <br/> <center> + <img src="w100.png" alt="Figure 1" style="width: 70%;" /> </center> @@ -74,16 +84,18 @@ We first use laser printing to create a mask consisting of five layers of chips <br/> - +</font> ## **Test of trap chamber** +<br/> + We tested the feasibility of the chip and proved that our chip can bind E. coli in the trap chamber. We used a 60x microscope to take time series images of E. coli activity in a trap [Figure 4], and found that E. coli was bound in the trap, and we could take clear and stable growth images. <br/> -<center><img src="/r2/chip.gif" alt="Figure 1" style="width: 70%;" /></center> +<center><img src="/r2/chip.gif" alt="Figure 1" style="width: 40%;" /></center> <font size=3 color=grey> @@ -95,7 +107,9 @@ We tested the feasibility of the chip and proved that our chip can bind E. coli <br/> -## **Test of oxygen concentration** +## **Test of oxygen concentration** + +<br/> We have tested the feasibility of the chip and proved that the chip can form an oxygen concentration gradient. The measurements were made using the ruthenium tris(2,2 '-dipyridyl) dichloride hydrate (RTDP) indicator. We loaded RTDP into the culture layer. The loading layer only load air at first, and then load nitrogen and air at the same time after a period of time to obtain the change of fluorescence intensity of the six channels over time [Figure 5]. Due to the defects of the air pump, the concentration gradient is not stable, but after standardization, there is a difference in fluorescence intensity between the six channels [Figure 7], which indicates that our chip does construct six different oxygen concentration environments. @@ -108,7 +122,7 @@ Due to the defects of the air pump, the concentration gradient is not stable, bu <br/> -<center><img src="w9.png" alt="Figure 1" style="width: 70%;" /></center> +<center><img src="hard-fig5.jpg" alt="Figure 1" style="width: 70%;" /></center> <font size=3 color=grey> @@ -139,8 +153,10 @@ $$I_0/I=1+K_q [O_2]$$ <br/> -## Conclusion +## **Conclusion** + +<br/> We design and successfully construct a microfluidic chip, and detect the chip's ability to trap Escherichia coli and to facilitate the creation of oxygen concentration gradients. Due to time constraints, we have not used the chip for measuring bacterial fluorescence intensity, but this chip is proven to be feasible. Our chip also allows for experimental measurements of expression variation caused by different lactate concentrations in the culture medium. -Our chip has a wide range of application scenarios where fluorescence measurements targeting E.coli can be conducted on it, while enabling precise control over oxygen concentration and culture medium concentration. +Our chip has a wide range of application scenarios where fluorescence measurements targeting E.coli can be conducted on it, while enabling precise control over oxygen concentration and culture medium concentration. \ No newline at end of file diff --git a/pages/education.vue b/pages/education.vue index 16c8abec9a494a732b031baf2024c271b6be53bd..e4d6cc0aed826d6cd2498e822f4271f5ff15f915 100644 --- a/pages/education.vue +++ b/pages/education.vue @@ -264,14 +264,14 @@ played games related to finding primers and understanding the principles of restriction enzymes. We also promoted the iGEM competition, and the students had a highly positive response to these activities. <center> - <img src="https://static.igem.wiki/teams/4713/wiki/site-figure/college.jpg" alt="fig3" style="width: 50%" /> + <img src="https://static.igem.wiki/teams/4713/wiki/edu/lei.jpg" alt="lei" style="width: 50%" /> </center> We also organized lectures on thanatology to help students in the School of Life Sciences recognize the importance of the 'life and death' issue. This aimed to ensure that they do not become solely absorbed in a technocratic mindset. In addition to enriching their sense of life's meaning, they will also better respect the lives of experimental subjects in future research. <center> - <img src="https://static.igem.wiki/teams/4713/wiki/edu/lei.jpg" alt="lei" style="width: 50%" /> + <img src="https://static.igem.wiki/teams/4713/wiki/site-figure/college.jpg" alt="fig3" style="width: 50%" /> </center> We also collaborated with six other universities to organize a popular science conference on microbiome-based cancer therapy. This allowed more students to experience the applications of synthetic