Team:Manchester/Contribution
CONTRIBUTION
Manchester iGEM 2019 may be exploring new horizons, looking into new applications of bacteria to industrial processes - but we are not doing it alone. Throughout our iGEM journey, we have questioned our peers and be questioned by them, taking part in a free exchange of idea we documented below. From giving a nudge to human practices through survey questions, to writing specialist parts of a wiki guide (Collaborations, Act II: Edinburgh - Remedye; Act VI: US AFRL Carroll HS) iGEM Manchester 2019 has spread the Cutiful vibes around the world, in the purest iGEM spirit.
Our achievements …
In 3 bullet points
1.
Characterised many Parts -Characterised BBa_J23102 (strong constitutive promoter), BBa_J23106 (weak constitutive promoter), and BBa_K199118 (T7 promoter-B0034-mRFP1) by measuring Relative Fluorescence Units (RFU) (Excitation (Ex) ƛ 574, Emission (Em) ƛ 618) and normalising it with Optical Density (OD) at 600 and 660 nm.
2.
Red fluorescence -Determined the importance of using OD660 for red fluorescence protein measurements.
3.
Characterised expression -Characterised and Compared the above BioBricks with pTetR-B0034-mRFP1 (BBa_K092300; alternative backbone = pBbB2c which has low copy number).
AIM
We wanted to add new, normalised RFU data for two promoters from the Anderson family of constitutive promoters BBa_J23102 (Strong constitutive promoter) and BBa_J23106 (weak constitutive promoter) respectively; and a T7 promoter BBa_K199118 all expressing mRFP1. We additionally wanted to compare our results with a fourth promoter, TetR (BBa_K092300). TetR promoter was cloned into a different vector, pBbB2c and also governed expression of mRFP1. Another interest that we had was to test how OD was affected by RFP production, therefore we performed our measurements twice, at OD600 and OD660 respectively. This was done because it has been shown that OD660 gives a more accurate representation of bacterial growth in RFP-producing bacteria (Hecht et al., 2016).
Table 1. Depicts the BioBrick structure and the iGEM registered code. When appropriate it also provides information as to its positioning on the iGEM 2019 kit plate as well as the vector, copy number and resistant maker gene (Knight, 2004; Morgan, 2014).
| Name | Biobrick | 2019 iGEM kit plate location | vector and copy number | Resistant marker gene |
|---|---|---|---|---|
| BBa_J23102 | 17G (6) | BBa_J61002~15-20 copies per cell | Carbenicillin | |
| BBa_K199118 | 17O (6) | BBa_J61002~15-20 copies per cell | Carbenicillin | |
| BBa_K199118 | 9M (4) | pSB1C3100-300 copies per cell | Chloramphenicol | |
| BBa_K092300 | Not provided | pBbB2c 20 per cell | Chloramphenicol |
Act II: METHODS:
Initially, we analysed the iGEM provided constructs and BBa_K092300 in DH5⍺ only through their mRFP1 expression. Those plasmids were transformed into E. coli DH5⍺ and BL21(DE3). However, note that BBa_K199118 was transformed into BL21(DE3) cells only, as DH5⍺ lacks the T7 polymerase and no colour production would be expected. Samples were then plated on antibiotic-containing agar plates (See Table 1). The plates were left incubating overnight at 37ºC. The following day a single colony from each plate was picked and inoculated in 5 mL of LB, then it was placed at 37ºC with shaking at 180 rpm for overnight growth. The following morning this sample was re-inoculated in a 1:100 dilution in LB media containing the required antibiotic. The cultures were grown at 37ºC with shaking at 180 rpm up to a desired OD600 of ~0.6. Next, BBa_K199118 was induced with 50 μM IPTG. 200 μL of each grown and induced culture was transferred onto a 4titude black, clear-bottom sterile 96-well plate. Of each sample 3 technical replicates were performed. Then, the plate reader (CLARIOstar®, BMG Labtech) was used to measure OD600 and RFU every 15 minutes overnight.
Following our initial experiment, we then repeated the same procedure, however, in this case the initial absorbance was measured at 600 nm for BBa_K199118 and BBa_K092300 and 660 nm for BBa_J23102 and BBa_J23106 before induction. This was done, as stated above, because OD 660nm provides a more accurate representation of bacterial growth in cultures expressing RFP (Hecht et al., 2016). Once the desired OD of ~0.6 had been reached, the required constructs were induced with 50 μM IPTG (BBa_K199118) and 100 nM of anhydrotetracycline (BBa_K092300) when required. After induction, samples were placed on a microplate reader (CLARIOstar®, BMG Labtech) and experiment with 3 technical replicates were performed. However, unlike our initial set of data in this case OD660 and RFU was measured every 15 minutes overnight. The data obtained was analysed following the same criteria as above.
Act III: RESULTS:
Quantitative results:
Below are the results we obtained for the OD and fluorescence measurements of the different promoters with mRFP1. All the values were analysed by blank-correction. For OD blank was LB media, and for RFU the blank was E. coli TOP10 since it does not express any colour. The values were individually normalised by dividing RFU/OD, and then averaged to plot the mean against time. An RFU value of 0 corresponds to baseline E. coli TOP10 measurements.
The plot shows the mean RFU/OD from three replicates of each construct expressed in E. coli DH5⍺. The OD was measured at 600 nm and RFP fluorescence was measured at Ex ƛ 574, Em ƛ 618, every 15 minutes for 13 hours. The RFU values were normalised by the OD and the triplicates averaged. All values have been blank-corrected. A total of 52 recordings were made per well, with three biological/technical replicates per construct.
The plot shows the mean RFU/OD from three replicates of each construct expressed in E. coli BL21 (DE3). The OD was measured at 600 nm and RFP fluorescence was measured at Ex ƛ 574, Em ƛ 618, every 15 minutes for 13 hours. The RFU values were normalised by the OD and the triplicates averaged. All values have been blank-corrected. A total of 52 recordings were made per well, with three wells per construct.
The plot shows the mean RFU/OD from three replicates of each construct expressed in E. coli DH5⍺. The OD was measured at 600 nm and RFP fluorescence was measured at Ex ƛ 574, Em ƛ 618, every 15 minutes for 13 hours. The RFU values were normalised by the OD and the triplicates averaged. All values have been blank-corrected. A total of 52 recordings were made per well, with three wells per construct.
The plot shows the mean RFU/OD from three replicates of each construct expressed in E. coli BL21 (DE3). The OD was measured at 600 nm and RFP fluorescence was measured at Ex ƛ 574, Em ƛ 618, every 15 minutes for 13 hours. The RFU values were normalised by the OD and the triplicates averaged. All values have been blank-corrected. A total of 52 recordings were made per well, with three wells per construct.
Qualitative results:
Non-quantitative data of the iGEM-provided coloured constructs were also obtained through the pelleting of 5 mL of overnight induced culture at 10000 xg for 10 minutes. These pellets were then transferred to 2 mL Eppendorfs and spinned again at 19, 900 xg in a conventional table-top microcentrifuge and imaged as shown (Figure 1).
RFP weak constitutive promoter
mRFP1 with a T7 promoter
Figure 1. 5 mL overnight bacterial culture pellets. In both BBa_J23102 and BBa_J23106 replicates 1 and 2 were transformed into BL21(DE3) (left and centre) while replicate 3 was transformed into DH5⍺ (right). However, for BBa_K199118 all 3 biological replicates are shown in BL21(DE3). All Eppendorfs are shown under normal light (in white) and UV light (blue) for visualisation purposes only. Biological replicates of BL21(DE3) cells for T7 promoter.
This results do not visually show the difference in fluorescence between DH5⍺ and BL21(DE3). However, it is visible particularly for BBa_K199118 the difference in expression between the 3 performed biological replicates.
Act IV: CONCLUSION:
The normalisation of fluorescence measurements to OD showed that the constitutive promoters, as well as the T7 promoter, started with a very high fluorescence-to-OD ratio, which decreased with time and then remained constant. This means that as the bacteria kept growing, the relative fluorescence measurements increased linearly with them. The strong constitutive promoter, J23102, showed the highest fluorescence-to-OD ratio followed by the weak constitutive promoter J23106 as we would expect and lastly the T7 K199118 which showed the lowest fluorescence-to-OD values despite possessing the highest plasmid copy number. This was true in both tested E. coli strains DH5⍺ and BL21(DE3). Additionally, this trend remained true in both OD600 as well as OD660 nm.
Data between OD600 and OD660 apparently varies between the two different tested E. coli strains. In DH5⍺, measurements at 600 nm are lower than measurements at 660 nm. Compared to BL21(DE3). Therefore we can conclude that a potential false increase in apparent cell density was only significant for the BL21(DE3) strain and not in the DH5⍺. This means that in our results, measuring optical density at 660 nm was only more appropriate for E. coli BL21(DE3) cells.
In conclusion, new normalised data for mRFP1 fluorescence has been characterised for three existing parts (BBa_J23102, BBa_J23106, and BBa_K199118) and for BBa_K092300 cloned into pBbB2c. This means that the relative fluorescence units can now be compared with higher accuracy and precision across measurements and even between different laboratories.
Act V: FUTURE EXPERIMENTS:
The increase of apparent cell density due to the presence of red fluorescent proteins was only observed in one of the two tested strains at OD660 . Therefore, maybe a higher optical density of 700 nm could allow a more accurate estimation of cell abundance in the presence of RFP.