Part:BBa_J364000

Test Device 1 for the iGEM InterLab Study

This is a GFP expressing constitutive device for the 2017 iGEM InterLab study. It is called Test Device 1 for the study for easy reference.

This device is stored in pSB1C3 for the InterLab and is fully BioBrick compatible.

Interlab 2022 Experiment 3 Device

• Test Device 1 for Experiment 3 of the 2022 iGEM Interlab

The following device is used in the iGEM 2022 InterLaboratory Study for Experiment 3.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 705
  

Team NAWI_Graz 2019: GFP dynamics of transformed Escherichia coli strains in LB broth

Background

In Team NAWI_Graz 2019, we found that in normal growth/incubation condition (37^oC, LB media + chloramphenicol) BBa_J364000-transformed Escherichia coli BL21 cells had the highest GFP expression in comparison to BL21 Star and Top 10 strains. Values were consistently higher for BL21 strain over the entire 44-hours measuring period.

Figure 1: GFL Flourescence Measurements with GFP containing E.coli strains and non GFP containing E.coli strains (control)). Remark: the green line represents all the GFP empty strains

Experimental Design

We used three different strains of transformed E. coli (Top10,BL21 and BL21 Star) for this study. Transformed strains were incubated in 15 mL LB broth 37 ^oC overnight. Next day they were used to inoculate a 50 mL LB to OD₆₀₀=0.1. The cultures were then incubated at 37 ^oC, 140 rpm and samples were taken every 4 hours for 2 days to determine the green fluorescence protein (GFP) fluorescence intensity at 510 nm.

Result and Findings

• There are significant differences of GFP expression in different strains of E. coli (Top10, BL21, BL21 Star)
• Cultures turned light green under white light after around 14-18 hours of incubation time.

          Figure 2: GFL Flourescence and OD600 Measurements with GFP containing E.coli strains and non GFP containing E.coli strains (control))

Figure 3: at the 14h time point old GFP-transformed E. coli strains (Left: Top10, Middle: BL21, Right: BL21 Star)

Improvement

• Group: iGEM Team UESTC-China 2019
• Author: Qiuyun Huang, Zixin Wang

---

This year, we improved this reporter device (BBa_J364000) into a surface display system (BBa_K3034007) (Fig. 1) by fusing GFP with INPNC (BBa_K523008). The improved system can anchor the downstream protein of INPNC to the surface of bacteria, while the GFP is used as the reporter gene. So, other teams who have the need of surface display can insert their target gene into this system.

Fig.1. Schematic map of BBa_K3034007.

Ice nucleation protein (INP) is a secretory outer membrane protein from Pseudomomas syringae, P.flurorescens and several other Gram-negative bacteria. INP can anchor one or more "passenger proteins" to the outer membrane of bacteria. The fixation of exogenous proteins on the bacteria surface through INPNC can not only greatly improve the efficiency of enzymatic reaction, but also avoid the degradation of exogenous proteins by intracellular enzymes of host cells[1].

Besides, we added a segment of linker between INPNC and GFP to ensure that two adjacent domains do not sterically interfere with one another. In our experiments, without linker, GFP could not be properly expressed.

Quantitative detection of fluorescence

First, we cultured the bacteria overnight and adjusted them to the same OD600. We ultrasonic broke, centrifuged and respectively resuspend precipitation to measure the fluorescence intensity of GFP in E.coli DH5α carrying BBa_J364000 and E.coli DH5α carrying BBa_K3034007 (Fig. 2).

Fig.2. The relative fluorescence intensity of E.coli DH5α carrying BBa_J364000 and E.coli DH5α carrying BBa_K3034007. The relative fluorescence intensity= Fluorescence of precipitation/ (Fluorescence of supernatant+ Fluorescence of precipitation)×100%.

The results showed that both precipitation and supernatant contained relatively strong GFP. Moreover, the distribution of GFP in E.coli DH5α carrying BBa_K3034007 was not significantly different from that in E.coli DH5α carrying BBa_J364000. There are some differences with our expectation, after analysis, it may be caused by incomplete ultrasonic broken of bacteria.

Since the E.coli DH5α carrying BBa_K3034007 expressed GFP, this indirectly indicated that INPNC was successfully expressed. However, the content of GFP in the E.coli DH5α precipitate (cell membrane) carrying BBa_K3034007 was not significantly higher than the E.coli DH5α carrying BBa_J364000. We hypothesized that INPNC was expressed but the efficiency was not so high.

Microscopic observation

Next, we used fluorescence microscopy to see if the INPNC worked. E.coli DH5α carrying BBa_J364000 (GFP) was rod-shaped and the fluorescence was equably distributed in E.coli (Fig. 3a). The fluorescence of E.coli DH5α carrying BBa_K3034007 (INPNC+GFP) was observed to be dotted and dispersed on the surface of E.coli (Fig. 3b,3c). The results proved that GFP has apparently been anchored to the surface of the E.coli and INPNC was working.

In addition, we also noticed that E.coli DH5α carrying BBa_K3034007 (INPNC+GFP) had fluorescence aggregation on one side of the E.coli surface (Fig. 3c). The result is consistant with fact that we found in the literature[2] that the INPNC forms aggregates in the cell membrane.Thus, we are more clear that we have achieved our improvement.

Fig.3. The fluorescence microscopy of E.coli DH5α carrying BBa_J364000(a) and E.coli DH5α carrying BBa_K3034007 (b、c).

Conclusion

1. We improved this part. GFP is an intracellular reporter gene, and we achieved the expression of GFP on the surface of E.coli.
2. Based on the current results, there is space for further improvement in the efficiency of system expression, and the key point is that the activity of INPNC needs to be improved.
3. Other teams who have the need of surface display can insert their target gene into this system. For example, the system can be applied to whole-cell biocatalysts, heavy metal contamination adsorption, antigen and antibody display, etc[1].

Team PuiChing Macau 2021: E.coli growth in food waste analysis

We would like to analyze the growth of E.coli via Green Fluorescent Protein (GFP). To examine the growth of E.coli in food waste, the Optical Density of E.coli is commonly used. Yet, in the presence of food waste (starch from rice as an example here), it is difficult to measure the O.D. value of E.coli growth at a wavelength of 600 nm (O.D.600), where determining the growth phase is almost impossible. Thus, we would like to use Green Fluorescent Protein (GFP) (488 nm and 510 nm) to establish a model of E.coli’s growth in food waste (starch). ====Result==== We estimate the number of bacteria in the culture by assessing the emission wavelength for fluorescence signals at O.D.510 and O.D.600. The tested samples are composed of 100ml of LB medium and 1.5ml of overnight bacteria culture. The time points measured are 0hr, 4hr, 12hr, and 24hr. As demonstrated by Fig.3 (3a, 3b, 3c, 3d), samples with GFP have shown higher absorbance compared with controls of BL21. And samples in the presence of food waste (starch) have also shown higher absorbance than those without food waste (starch).

Fig 1a, 1b. Experiment culture (bacteria and GFP) centrifuged: 1a normal; 1b under UV light(from left to right: 1. LB 2. LB+starch 3. GFP+starch 4. GFP 5. BL21+starch 6. BL21

Fig 2a 2b. Experiment culture (bacteria and GFP) under UV light: 2a with starch; 2b without starch (from left to right: 1. BL21+starch 2. GFP+starch 3. GFP 4. BL21)

Fig 3a O.D.600 of BL21 culture with starch and without starch

Fig 3b GFP signal intensity of BL21 culture with starch and without starch

Fig 3c O.D.600 signal intensity of BL21 culture with starch and without starch (normalized)

Fig 3d GFP signal intensity of BL21 culture with starch and without starch (normalized)