Difference between revisions of "Part:BBa B0030"

Group: QHFZ-China iGEM 2019
Author: Cheng Li
Introduction & Design:
This year we use BBa_B0030 to construct BBa_K3007010 (https://parts.igem.org/Part:BBa_K3007010). We designed a gene circiut that can expressed sfGFP or dsRed under the induction of uric acid. RBS0030 (BBa_B0030) is a strong RBS and we used it to powerfully promote the expression of fluorescent proteins.

Documentation:
We charactered RBS0030 part in the part BBa_K3007010 (https://parts.igem.org/Part:BBa_K3007010). Fig.4 and Fig. 5 showed the result. Prin_p80α and RBS0030 controled the expression of sfGFP. Fig. 1-3 showed the process that we constructed the part.

This year, QHFZ-China designed a UA monitor system in Escherichia coli (E. coli). The original version is shown in Fig. 1. Pc is a constitutive promoter, Pcp6 promoter, and it promotes the expression of HucR and YgfU. When uric acid is absent, HucR can bind to PhucR, which suppresses dsRed expression. If uric acid presents in high concentration, HucR will release from PhucR and the expression of dsRed will recover from the inhibition [2].

Two clones with the UA detection system were tested. The original gene circuit was able to response to UA in a range of 0 to 200 μM (Fig. 2A). The clone 1 showed much better dynamics than the other (Fig. 2B). Time course experiments showed that the fluorescence intensity became quite strong at 4 to 6 hours after UA induction, and became stable at 10 to 12 hours (Fig. 2C). Even if we removed UA by replacing fresh LB medium, after 48 hours shaking, the fluorescence would be still notable (Fig. 2D) and there was not significant difference of dsRed fluorescence / OD600 between before and after UA removing (Fig. 2E). All the data meant our design could detect high UA concentration quickly and stably.

However, through our human practices, we found the sensitivity and responding time of the original design are not good enough. In the next generation design, we introduced RinA_p80α - PrinA_p80a system to enhance the sensitivity. Meanwhile, we changed dsRed to sfGFP, whose maturation time is much shorter, to shorten the waiting time. The new version of the uric acid detector was shown in Fig. 3. If UA presented, RinA_p80α would express and active transcription of sfGFP which was under control of PrinA_p80α. We called this as Version 2.

We tested the sfGFP production of Version 2 under different concentration of extracellular UA. The curve in Fig. 4A showed the fluorescence was saturated under only 15 μM UA induction, while the old version needed about 100 μM UA to get saturated (Fig. 2B). To test if sfGFP could shorten the reaction time, we used the same construct only except reporter genes, called PRinA_p80α – sfGFP and PRinA_p80α – dsRed, respectively. After adding 20 μM UA into the reaction system, the curve of PRinA_p80α – sfGFP climbed much faster than PRinA_p80α – dsRed, which suggested our new design had a great induction performance, and fitted our predictions very well (Fig. 4B).

At last, we took a photo to show the green fluorescence released by E. coli expressing sfGFP.

References:
[1] Wan, X., Volpetti, F., Petrova, E., French, C., Maerkl, S. J., & Wang, B. (2019). Cascaded amplifying circuits enable ultrasensitive cellular sensors for toxic metals. Nature chemical biology, 15(5), 540.
[2] Liang C., Xiong D., Zhang Y., Mu S. and Tang S. (2015). Development of a novel uric-acid-responsive regulatory system in Escherichia coli. Appl. Microbiol. Biotechnol. 99, 2267–2275.

Contribution: WHU-China 2019

pFadD_Lac is a fatty acid-sensitive promoter, one of the regulators in the enzymes of fatty acid biosynthesis in E. coli. It is composed of two fadR recognition sites. In the absence of fatty acids, an intrinsic transcription factor FadR acting as the repressor in the fatty acid metabolism system in E. coli. will attach to the promoter, and block the downstream gene expression. This has been proved to be effective by a previous iGEM team（NTHU_Taiwan）. [1][2] 2019 NTHU_Taiwan further modified pFadD promoter by replacing its CRP binding site with a LacI repressor binding site, which reduced expression leakage. We combine the RBS(B0030) with the promoter pFadD_Lac which is sensitive to fatty-acids and use GFP to test the performance of the promotor by measuring the fluorescence intensity.

Moreover, according to the fact that most RBS with higher binding efficiency had the repetitive sequence of "AGGG", or "AAA" after the start base, we improved a new part of RBS and tried to compare it with the original RBS (BBa_B0030) to see which is better in some specific occasions.

After the successful transformation of the plasmid, we used different concentrations of fatty acids for induction for different times. The expression level of eGFP was determined by fluorescence detection with a microplate reader. The results are as follows:

According to the results, we can find that the performance of our fatty acids-sensitive promoter is improved to a certain extent. Taking 0.125× fatty acid concentration as an example, the fluorescence intensity of BBa_K3763003 is over 11000 after 6 hours, while the promoter of BBa_K3763002 is less than 10000 at three fatty acid concentrations. However, we can see that the optimized promoter still has a high expression leakage and low expression in a short time. However, the experimental results have been able to prove the function of the fatty acids-sensitive promoter, and partially prove the effectiveness and rationality of our design.

Reference [1] https://2019.igem.org/Team:NTHU_Taiwan/Results [2] http://2012.igem.org/Team:NTU-Taida#

Functional Parameters: Austin_UTexas

BBa_B0030 parameters

Burden Imposed by this Part:

Burden is the percent reduction in the growth rate of E. coli cells transformed with a plasmid containing this BioBrick (± values are 95% confidence limits). This BioBrick did not exhibit a burden that was significantly greater than zero (i.e., it appears to have little to no impact on growth). Therefore, users can depend on this part to remain stable for many bacterial cell divisions and in large culture volumes. Refer to any one of the BBa_K3174002 - BBa_K3174007 pages for more information on the methods, an explanation of the sources of burden, and other conclusions from a large-scale measurement project conducted by the 2019 Austin_UTexas team.

This functional parameter was added by the 2020 Austin_UTexas team.

B0030 vs B0034 in E.coli BL21 (DE3) - Thessaly 2024

Group: Thessaly 2024

Purpose
B0030 and B0034 were the RBSes that we used in our promoter testing experiments. We wanted to check which RBS would be the strongest and which would be the ideal candidate for our design.

Methods
Measurements for OD 600nm and fluorescence (488nm,510nm[1]) were taken over the course of 16 hours, in a 96-well microplate (black plate, clear bottoms). Clonings were done according to the Golden Braid method, leaving us with level 1 constructs in the pDGB3a1 backbone ( BBa_K4213058. )

Construct transformed into E.coli BL21 (DE3) chassis, incubated at 37oC, 180rpm for 16 hours.

Here you can find more information about the constructs tested containing BBa_B0030: BBa_K5299200, BBa_K5299202,BBa_K5299204.
Here you can find more information about the constructs tested containing BBa_B0034: BBa_K5299201, BBa_K5299203,BBa_K5299205.

Plated 120 ul 5 times, out of each single liquid bacterial culture, created from the same bacterial colony, in order to establish accuracy through technical repeats.

Medium used was M9 due to minimal interference, with D-glucose serving as the carbon source. Also, served as blank, plated 5 times.

Measurements were normalised as such: using the average price of fluorescence for the 5 technical repeats and dividing it by the average price of OD.
Standard deviation included in the graphs.

Results
Measurements were taken over the course of 16 hours.Here are the results for BBa_B0030 regarding BBa_J23119 with the pDGB3a1 backbone. BBa_B0030 proved less strong than BBa_B0034.

Figure 1: Constructs with the BBa_J23119 promoter, the BBa_B0030 or the BBa_B0034 RBS, the BBa_I746916 sfGFP and the BBa_B0015 terminator. The pDGB3a1 backbone served as a negative control.

For more information regarding BBa_J23119, head over to BBa_J23119.

Here are the results for BBa_B0030 regarding BBa_J45992 in the pDGB3a1 backbone. BBa_B0030 proved less strong than BBa_B0034.

Figure 2: Constructs with the BBa_J45992 promoter, the BBa_B0030 or the BBa_B0034 RBS, the BBa_I746916 sfGFP and the BBa_B0015 terminator. The pDGB3a1 backbone served as a negative control.

For more information regarding BBa_J45992, head over to BBa_J45992.

Here are the results for BBa_B0030 regarding P3.1 (BBa_K4583008) in the pDGB3a1 backbone. BBa_ B0030 proved less strong than BBa_B0034.

Figure 3: Constructs with the P3.1 promoter, the BBa_B0030 or the BBa_B0034 RBS, the BBa_I746916 sfGFP and the BBa_B0015 terminator. The pDGB3a1 backbone acted as a negative control.

For more information regarding P3.1, head over to BBa_K4583008.

References

[1] Pédelacq, J.-D., Cabantous, S., Tran, T., Terwilliger, T. C., & Waldo, G. S. (2006). Engineering and characterization of a superfolder green fluorescent protein. Nature Biotechnology, 24 (1), 79–88. doi:10.1038/nbt1172