Regulatory
Ara

Part:BBa_K808000:Experience

Designed by: Valentina Herbring, Sebastian Palluk, Andreas Schmidt   Group: iGEM12_TU_Darmstadt   (2012-08-11)


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iGEM NU Kazakhstan 2024 characterization

AraC-pBAD

AraC-pBAD part consists of the pBAD promoter sequence and L-arabinose regulator AraС, which coding sequence is transcribed in the opposite direction [1]. AraC is induced in the presence of L-Arabinose and inhibited when glucose is added. In the presence of glucose, cellular cAMP levels decrease, preventing the cAMP-CRP complex from binding to the pBAD promoter, thereby repressing the promoter's activity. In the absence of L-arabinose, AraC forms a loop in the promoter region, blocking transcription. When L-arabinose is bound to the AraC-pBAD promoter, the conformation of AraC changes resulting in the induction of transcription [2]. Since L-arabinose is non-toxic and relatively inexpensive sugar, the application of the AraC-pBAD system is affordable and highly versatile for various genetic and metabolic applications. In order to test the response of AraC-pBAD to the induction with L-Arabinose, E.coli cells transformed with the plasmid containing MazF ribonuclease (Kill Switch) coding sequence under AraC-pBAD promoter were incubated with various concentrations of arabinose (0.05%, 0.1%,0.2%, 0.5%, 1%). Since MazF acts as a toxin for the cells producing the endoribonuclease via cleaving mRNA molecules and subsequently inhibiting protein synthesis, cells synthesizing MazF ribonuclease will eventually die.

OD600 nm values of transformed E.coli cells
Figure 1. OD600 nm values of transformed E.coli cells incubated with different concentrations of arabinose.


Figure 1 summarizes the OD600 nm values of transformed E.coli cells after incubation with 0.05%, 0.1%,0.2%, 0.5%, 1% arabinose. The results indicate that even 0.05% concentrations of arabinose led to the expression of AraC-pBAD promoter which was observed by a significant decrease in OD600 nm values caused by MazF expression (x̄ for the control untreated group = 1.57, x̄ for 0.05% ara group = 1.08). As arabinose concentration gradually increases from 0.05% to 1%, a decline in the vitality of the samples is observed. The lowest proliferation level was recorded at 1% arabinose, indicating that this concentration of arabinose resulted in the highest expression of the AraC-pBAD promoter and subsequent MazF synthesis.

In the control group that was not treated with arabinose, high proliferation of E.coli cells was observed, thereby MazF protein was not expressed. The obtained results indicate that without arabinose the AraC-pBAD promoter has very low basal expression levels and leakage, which makes it suitable for expression of highly toxic proteins such as MazF.

iGEM TU Darmstad-2012 team measured the induction of AraC-pBAD promoter with different arabinose concentration using GFP. The results of induction showed that cells exhibited fluorescence after incubation in a medium containing more than 0.01% (w/v) arabinose, thereby confirming promoter expression even at low arabinose concentrations [1].

To assess promoter leakiness, iGEM TU Darmstad-2012 team amplified construct containing AraC-pBAD promoter (BBa_K808000), 15b-5p toehold switch (BBa_K2206000), and GFP coding sequence (BBa_E0040). They extracted plasmid DNA containing the construct and sequenced it [1]. The results indicated successful amplification without fidelity errors and toxic levels of 15b-5p toehold switch which shows that AraC-pBAD promoter has low leakage.

However, when iGEM TU Darmstad-2012 team performed amplification with the plasmid DNA containing toehold switch for hsa-miR-27b-3p (BBa_K2206007) under AraC-pBAD promoter (BBa_K808000), the results indicated the presence of mutations in promoter and production of toxic levels of BBa_K2206007 without induction of promoter [1]. In this experiment, the AraC-pBAD promoter was leaky.

Additionally, AraC-pBAD promoter was characterized by iGEM Groningen-2019 team. The team measured inducibility of the promoter in V. natriegens at 0.01, 0.05, 0.1 and 0.5% arabinose concentrations and found that activity of promoter started at 0.05% arabinose concentration and highest expression was observed for 0.5% arabinose concentration. The team also confirmed a low basal level of expression without arabinose, indicating low leakage.


References

[1] iGEM Parts Registry. (2012). Part: BBa_K808000. https://parts.igem.org/Part:BBa_K808000

[2] VectorBuilder. (n.d.). pBAD Bacterial Recombinant Protein Vector https://en.vectorbuilder.com/resources/vector-system/pBAD.html

USP-Brazil 2019 Characterization

Methods

In our project we aimed to compared 3 different promoters (pBad, pLac and pT3). For analysing the promoters, we choose to utilise the characterization reporter (BBa_K2771020) because it already had the reporter gene (eYfp) for the quantification of expression, and a constitutive reporter (eCfp) for normalization of measurements. Utilise this reporter allows us to have a better compassion between different promoters, since has the same "reporter backbone" the comparison is normalised by the eCfp, providing a more accurate comparasion. As shown in the Figure 1, the 3 plasmids were correctly constructed.

T--USP-Brazil--Promotercomp1.png Figure 1: (A) – Colony PCR: an amplification of about 2000 bp fragment confirms the insertion of pT3 (lane 3). (B) – Colony PCR: a fragment of about 3300 bp confirms the insertion of pLac (lanes 1-3) and pBAD (lane 9). Red arrow indicates the chosen colonies for induction assay.

Results

Because LB broth media has a natural fluorescence that hinders the measurement of reporter proteins, we decided to use a Minimal Medium supplemented with Leucine and Vitamin B1, as our strains (DH10B and HST08) were respectively auxotrophic for these components. The carbon sources used for this experiment were Glucose or Glycerol.

The ratiometric promoter characterization reporter + pLac or pBAD constructs were cloned in DH10B strain, while ratiometric promoter characterization reporter + pT3 construct was cloned in HST08 carrying single blue light sensor (BBa_K3095003). The following graphs (Figure 2) show the experiment performed with pLac and pBAD constructs. Unfortunately we did not get any results for pT3 construct, since it took a lot of time (still taking) to standardise this assay.

T--USP-Brazil--Promotercomp2.png Figure 2: Expression of yfp induced by IPTG or (L)-Arabinose with Glucose or Glycerol as carbon source. –Control is DH10B strain carrying characterization reporter (BBa_K2771020) without promoter for yfp expression.

From the induction experiment we could contribute with accurate data by plotting our graphs with normalised expression of the reporter. Also, we compared the induction between Glucose or Glycerol as carbon source. As we can see in the graphic, the pLac showed a very higher expression then the pBad. However a normalisation of the inducer molecule is necessary for a more accurate analysis.

Applications of BBa_K808000

User Reviews

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DTU-Denmark

We experienced the promoter to be very leaky. We improved the part and the new promoter is considerably more tight (Part:BBa_K1067007) also see the library of different pBAD promoters that can be use to improve and model BBa_K808000.

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CLSB-UK

Overview:

We aimed to test our constructs in a cell free system following amplification in E.coli. However, we found that the transcripts from our constructs were produced in toxic concentrations to E.coli under the control of the constitutive promoter BBa_J23111. Thus, to prevent transcription during amplification, we made new constructs containing the promoter BBa_K808000, as it was reported to have very low leakage. We characterised BBa_K808000 in a cell free system by measuring the amount of GFP produced in response to varying concentrations of arabinose. We also determined if this promoter is suitable for regulating the expression of lethal parts in E. coli.

Methods:

For characterisation of the leakage of the promoter, we amplified our constructs containing BBa_k80800 in E.coli and performed plasmid minipreps to extract circular DNA for expression in a cell free system. We then sequenced our plasmid DNA.

For characterisation of BBa_K808000 in response to arabinose concentrations, we prepared a cell free system containing BBa_K808000 from the parts registry and left it to incubate for 10 hours at 37°C. The cell free system we used was the E.coli S30 extract system for circular DNA from Promega. We then added 1 μl of arabinose at the concentrations of: 0.05%, 0.1%, 0.5%, 1% and 2% and ran the experiment for 10 hours at 37°C, recording fluorescence intensity every 10 minutes.

Results:

Characterisation during amplification of our constructs:

Sequencing analysis showed that we successfully amplified the part BBa_K2206006 (this part contains BBa_K2206000, BBa_E0040 and BBa_K808000) with no fidelity errors. Therefore BBa_K808000 was suitable for preventing toxic levels of our part BBa_K2206006 in E.coli, demonstrating that BBa_K808000 has low levels of leakage. However, we found some mutagenesis in the promoter (BBa_K808000) for part BBa_K2206007 (which contains BBa_K2206001, BBa_E0040 and BBa_K808000), meaning toxic levels of BBa_K2206007 were still produced. This indicates that the promoter has some leakage and may therefore be unsuitable for regulating the expression of lethal parts.

Characterisation using GFP:

We found that fluorescence increased in as little as one hour and maximum fluorescence was reached after ~8 hours for all the concentrations. We found that the fluorescence increase occurred with as little as 0.05% arabinose (we did not measure lower than this) and increased with arabinose concentrations up to 0.5%. Interestingly, we saw a decline in fluorescence at 1% and 2% arabinose concentrations.

This may be explained by the fact that in a cell free system there is a limited amount of nucleoside triphosphates (NTP) and ribosomes. At 1% and 2% arabinose concentrations we speculate that lots of GFP mRNA was produced in a short period of time. Consequently, the NTP pool was rapidly depleted and there was an excess of mRNA relative to the number of ribosomes. This led to degradation of the excess mRNA, which otherwise would have been produced later when there would be free ribosomes. Additionally, the excess production of mRNA in early stages resulted in only a few NTPs remaining for further mRNA production later on. Therefore, less mRNA was translated, so GFP production was reduced, leading to decreased fluorescence at higher concentrations.

05000100001500020000250003000035000400000100002000030000400005000060000700008000090000100000BBa_K808000 characterisation-ve control-ve DNA2%1%0.50%0.10%0.05%Time (seconds)Fluorescence (arbitrary units)


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