Team:SCU China/InterLab

InterLab Study

      We participated in this year’s InterLab work and uploaded the data and relative questionnaire. In this page, we will list materials and equipment used in the InterLab Study, explain the details of our method, which is described a bit briefly in the official protocol, show our results, and make a sincere acknowledgement to who helped us.

01  Materials and Equipment

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.

Material Name Resource
Competent cells Trans 5 alpha form
DNA 2017 kit plate 7
Tryptone OXOID LP0042
NaCl KELONG Company
Yeast extract OXOID LP0021
Water Aquae sterilisata
Agar Solarbio biotechnology grade
Chloramphenicol Solarbio USP grade
Ethanol CHRON chemicals
Parafilm BEMIS
plate Corning
Table 1. Details of experimental material

Equipment Name Resource
Thermostatic water-bath GUOHUA LKTC-B1-T
Fridge Midea BCD-610WKM(E) for -20 and 4 degrees centigrade, Haier biomedical ultralow temperature freezer for -80 degrees centigrade
Biosafety cabinet ThermoScientific 1300 series A2
Autoclave ZEALWAY GR85DA
Centrifuge ThermoScientific ligend micro 17
Thermostatic incubator HENGZI SPX-150-Z
Shaker ZHICHENG constant temperature incubator shaker
Plate reader/td> ThermoScientific Varioskan Flash
Table 2. Details of experimental equipment
02  Detail of Methods
Details of Transformation

Aquae sterilisata was used to dissolve the DNA powder in kit. After the certain DNA was dissolved, it was kept in -20 degrees centigrade fridge, as well as the DNA powder. When we prepared transformation experiment, competent cells and the DNA was taken out and defrosted on the ice. Mixture of DNA and competent cells was done out of the biosafety cabinet while adding of liquid LB was in it. Solid LB contained 2% agar and chloramphenicol was added before making plates. Parafilm was used to prevent the fast evaporation of plates in incubator.

Details of Measurement

      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.

Details of Preservation and Verification

      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.

03  Results

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

Plasmid Name Parts Name
Positive control (PC) J23151-B0032-E0040-B0010-B0012
Negative control (NC) R0040
Test device 1 (D1) J23101-B0034-E0040-B0010-B0012
Test device 2 (D2) J23106-B0034-E0040-B0010-B0012
Test device 3 (D3) J23117-B0034-E0040-B0010-B0012
Test device 4 (D4) J23101-J364100-E0040-B0010-B0012
Test device 5 (D5) J23106-J364100-E0040-B0010-B0012
Test device 6 (D6) J23117-J364100-E0040-B0010-B0012
Table 3. Name of plasmids and corresponded parts name

      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.

Fig. 1. Fluorescein Standard Curve and Its Log Scale
(Left) Fluorescein Standard Curve under ex485/em530; (Right) Fluorescein Standard Curve (log scale)

Fig. 2. OD600 Curve of InterLab Strains
NC and PC means the negative control and positive control respectively. Biological replicates were considered together and error bar shows the standard error of all the eight replicates.

Fig. 3. Fluorescent Strength Curve of InterLab Strains
Biological replicates were considered together and error bar shows the standard error of all the eight replicates.

Fig. 3. uM Fluorescein / OD600 Curve of InterLab Strains
Biological replicates were considered together and error bar shows the standard error of all the eight replicates.

      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.

04  Acknowledgement and Collaboration

      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).