Difference between revisions of "Team:SCU China/InterLab"

We did the experiment following the protocol provided on the iGEM official website, including the transformation procedure. Table 1 and table 2 show the details of our materials and equipment respectively.

      When we did the measurement. The cabinet was shined by ultraviolet before start for 30 minutes. One person (name C for convenience) took 500μl from each culture tube to a EP tube, then gave it to one person out of the cabinet (name B) to test the original OD600. Then, C prepared enough 50ml tubes with 12ml liquid LB in it (chloramphenicol preadded), and B calculated the dilution way according to the excel provided on official website. When the calculation was done, C piped out certain volume of LB and added bacterial culture, then shook to mix and took 500μl as the sample of 0 hour. After the first sampling, we took out the culture from incubator (220rpm, 37℃) every 2 hours, with the first sampling time denoting 0 hour.
      Plates with black frame and transparent, flat bottom were used to do the measurement. Since we did the experiment many times resulting from some accidents, we recycled the plates. Plates were cleaned by distilled water and 75% ethanol. Plate reader and relative software was manipulated by a teacher due to the requirement of the lab.

      After picking up single colonies, the plates were kept in -4 degrees centigrade fridge. Bacterial glycerol stocks were prepared with the final glycerol concentration being 25%.
      When we decided to use them, we first re-activated them by plate streaking, then picked single colonies for subsequent work. Sequencing using prime VF2 and VR was done by sending bacterial culture to Chengdu Tsingke zixi Limited Company. Sequencing results were aligned with sequences in iGEM Registry and all of them were right.

We successfully transformed the plasmids provided in 2017 kit plate 7. Name of plasmids and corresponded parts name are shown in Table 3. Short for name will be used to refer instead of parts name for convenience

      We did the measurement for several times after transformation. Since some mistake occurred at the beginning, we only uploaded the data of the last measurement. Here we show our result in figure 1, 2, 3, 4, representing for the fluorescein standard curve, curve of OD600, fluorescent strength and μM fluorescein per OD600 respectively. Data were calculated in the excel provided by iGEM official website. Blank control had been considered before plotting.

      According to these figures, we can conclude that in the aspect of promoter strength, J23101 is greater than J23106 and J23106 is greater than J23117, and in the aspect of RBS strength, B0034 is greater than J364100 (Figure 3, 4). Difference in RBS strength may be related to the length of spacer between SD sequence in RBS and start codon. We can also see that the growth of D1 and D4 were relatively influenced (Figure 2), suggesting J23101 may have a negative effect on bacterial growth, though the protein expression is relatively high.

      Caused by our carelessness, we put the Ludox in the -20 degrees centigrade and all of them precipitated. Here we sincerely acknowledge team “SiCAU-China” to provide us extra Ludox.       We sincerely gratitude the work done by some freshmen, Caiqin Wang, Tian Liu, Chenming Zhang, Yuanhan Yang, Yanling Zhong, Changhe Li (names listed in no particular order), in InterLab study.       We helped Team SCU-WestChina by giving them some of our bacterial glycerol stocks, and their feedback showed that our transformation was successful.       When we plotted the standard curve, we found that the fluorescent strength seemed lower than expected when fluorescence concentration is very high. Thus, we described this phenomenon in the WeChat group of iSWU for some suggestion. Thankfully, student leader of team “SiCAU-China” told us that that may be caused by the inner filter effect; that is to say, when the concentration of fluorescence is very high, the excitation light cannot fully excited all of them, causing the decrease of fluorescent strength. We also shared this point in CCiC (Central China iGEM Consortium).