Difference between revisions of "Part:BBa K3457003"

Latest revision as of 22:48, 21 October 2021

T7 promoter

T7 promoter of pet-modification vector

Contribution

Group: QHFZ-China iGEM 2020

This part is a basic part. QHFZ-China 2020 measured it in their composite parts, K3457032, K3457033, K3457034, K3457035, K3457036, K3457037, K3457039, K3457042, K3457043.

Improvement by iBowu-China 2021

In iGEM 2021, Team iBowu-China noted the 2020 QHFZ-China T7 promoter can not start the expression of sfGFP in its downstream. In our design, we adopted T7 promoters to express sfGFP or gusA sequence from E. coli and other species to express an enzyme β-glucuronidase. We tested the use of this "short" T7 promoter and noted a difference with a complete T7 promoter with four more bases added [1, 2]: the 2020 QHFZ-China T7 promoter can not start the expression of sfGFP in its downstream, while the complete T7 promoter with four more bases can. We further established, measured and documented the complete T7 promoter as an improvement on this 2020 part: T7 K3716007 ( K3716007 )。

Figure 1. Differences between the T7 promoter sequence by QHFZ-China-2020 and the T7 promoter sequence by iBowu-China-2020.

In Figure 1, the first promoter sequence is the old part from QHFZ-China 2020 (a short T7 promoter truncated), while the second T7 promoter (T7 consensus, iBowu-China 2021) has four more bases.

Introduction

T7 promoter is a commonly used promoter, which can be recognized by T7 RNA polymerase and efficiently transcribe downstream genes. This year we used this promoter to express sfGFP or β-glucuronidase enzyme in strains such as E. coli BL21 (DE3) to produce green fluorescence, or to convert glycyrrhizic acid into glycyrrhetinic acid. We used the T7 promoter part K3457003 established and measured by QHFZ-China 2020, but we found that this element cannot initiate downstream gene expression. Further research found that after adding four bases of GGAA to it, the function returned to normal. In fact we found that pet28a, a commonly used expression vector, has two connected features on its sequence, the T7 promoter and the LacO sequence, and these two sequences actually overlap. Specifically, the overlap is the four GGAAs at the end of the T7 promoter. The bases are also included in the LacO feature. We guess that QHFZ-China 2020 overlooked this problem when splitting these two elements, resulting in the lack of four necessary bases in the T7 promoter designed. In addition, according to the literature [1, 2], we have designed three other promoters, C4, H9 and G6. They can also be activated by T7 RNA polymerase, but the strength is different from that of T7 promoter, so that users can choose the right promoter according to the required expression strength.

Protocol

1. Transform the plasmids into E. coli BL21(DE3)
2. Pick a single colony by a sterile tip from each of the LB plates for all the experimental and control groups. Add the colony into 4ml LB medium with kanamycin. Add 1mM IPTG to all experimental groups as needed. Incubate at 37℃ in a shaker overnight.
3. Fluorescence. Acquire 100 ul bacteria culture and centrifuge at 10000g for 1 min. Aspirate the supernatant and resuspend with 100 ul ddH2O to get rid of the fluorescence background from LB medium. Add 100 µl sample solution into a sterile 96-well plate. Measure fluorescence with a microplate reader and then also measure OD600 for normalization.
4. SDS-PAGE.
   1. Take 1ml of bacterial solution, centrifuge at 12000g for 1min at 4℃, discard the supernatant, add 100ul of pre-cooled RIPA lysate (high), vortex to mix, settle for 5min, centrifuge at 4℃, 12000g for 10min, and take the supernatant which contains the protein extract. After protein quantification by BCA method using commercial test box, adjust the concentration of the protein solution to 1mg/ml. Take 4 parts of the protein solution and 1 part of 5X protein loading buffer and mix it in a boiling water bath for 5 minutes, centrifuge at 12000g at 4°C for 10 minutes, take the supernatant, and store on ice. Use precast gel purchased from commercial company (Transgen, Precast Tris-Glycine Gel) for protein electrophoresis.
   2. Add marker in the first lane on the left at 5μl, and add sample on all other lanes with 10ul sample on each lane. Use 100V for electrophoresis. After the electrophoresis, take out the gel, put it in the staining box, add Coomassie Brilliant Blue dye solution to about 3mm below the gel surface, shake on a horizontal shaker, dye for 30min at room temperature, discard the dye solution, wash off the floating color with distilled water, and add decolorization liquid, decolorize on a horizontal shaker until the result is in good condition for taking pictures.
5. Enzyme Activity
   1. Take 100 ul of bacterial solution, centrifuge at 10000g for 1min at room temperature and discard the supernatant. Resuspend with 100ul ddH2O, and add into this solution 100ul of standard test solution I (containing Phenolphthalein-β-D-glucuronide, and the test box was purchased from Nanjing Jiancheng Biotech company). Incubate the mixture at 37C for 1 hour.
   2. Observe the solution. pink or purple color indicates positive enzyme activity. Quantitatively the activity can be measured by OD540 reading on a microplate reader, divided by its OD600 reading for normalization of concentration.

Results

We added the above-mentioned promoters to the upstream of the sfGFP gene, and determine the intensity of each promoter by measuring its green fluorescence.

Figure 2. Green Fluorescence emitted by sfGFP from different promoters.

We also put the T7 promoter with GGAA in the upstream of the β-glucuronidase enzyme. Through SDS-PAGE, it can be found that this composite part can efficiently initiate the expression of the β-glucuronidase enzyme.

Figure 3. Expression of β-glucuronidase with Iptg induction, in the composite part including consensus T7 with GGAA tail. The red arrow marked the expected size band around 67 kDa where the enzyme is expected to be located. Control groups where there is no Iptg induction for the same plasmid and where the sequence was completely removed (labeled Pet) showed no intrinsic protein exists in the expected bands.

Further, the β-glucuronidase enzyme has been proved to show enzyme activity.

Figure 4. Measurement of enzyme activities and comparisons between the β-glucuronidase sequence with the full length T7 promoter (iBowu-China-2021 part) and a control group (vacant-pet).

Summary

T7 K3457003 does not have the ability to initiate transcription. T7 part K3716007 ( K3716007 from iBowu-China-2021), which has been added with a tail of 4 bases GGAA, has this ability. Several other promoters mutated from this T7 promoter can also initiate transcription, but the strengths are different and not consistent with those reported in the literature [1, 2]. You can choose a promoter reasonably according to the needs of expression intensity and strength.

References

[1] Jones, J., Vernacchio, V., Lachance, D. et al. ePathOptimize: A Combinatorial Approach for Transcriptional Balancing of Metabolic Pathways. Sci Rep 5, 11301 (2015). https://doi.org/10.1038/srep11301

[2] Adams, A. M., Kaplan, N. A., Wei, Z. et al. In vivo production of psilocybin in E. coli. Metabolic Engineering, vol 56 p111-119, (2019). https://doi.org/10.1016/j.ymben.2019.09.009.

Sequence and Features