Part:BBa_C0012

lacI repressor from E. coli (+LVA)

Coding region for the LacI protein with an LVA degradation tail and without an RBS. LacI binds to the pLac regulator BBa_R0010 and PLlac01 hybrid regulator BBa_R0011 and inhibits transcription. [http://openwetware.org/wiki/IPTG IPTG (Isopropylthiogalactoside)] binds to LacI and inhibits its operation, therefore promoting transcription.

A rapid degradation tail (LVA) has been added to improve the switch time for High to Low performance of this part.

Usage and Biology

This particular LacI protein was derived from E. coli and contributed by Michael Elowitz. (See Part Design for more information.)

Image from the Protein Data Bank (PDB - 1LBI)

• • Allergen characterization of BBa_C0012: Not a potential allergen

The Baltimore Biocrew 2017 team discovered that proteins generated through biobrick parts can be evaluated for allergenicity. This information is important to the people using these parts in the lab, as well as when considering using the protein for mass production, or using in the environment. The allergenicity test permits a comparison between the sequences of the biobrick parts and the identified allergen proteins enlisted in a data base.The higher the similarity between the biobricks and the proteins, the more likely the biobrick is allergenic cross-reactive. In the full-length alignments by FASTA, 30% or more amount of similarity signifies that the biobrick has a Precaution Status meaning there is a potential risk with using the part. A 50% or more amount of identity signifies that the biobrick has a Possible Allergen Status. In the sliding window of 80 amino acid segments, greater than 35% signifies similarity to allergens. The percentage of similarity implies the potential of harm biobricks’ potential negative impact to exposed populations. For more information on how to assess your own biobrick part please see the “Allergenicity Testing Protocol” in the following page http://2017.igem.org/Team:Baltimore_Bio-Crew/Experiments

For the biobrick Part:BBa_C0012, there was a 0% of identity match and 0% similarity match to the top allergens in the allergen database. This means that the biobrick part is not of potential allergen status. In 80 amino acid alignments by FASTA window, no matches found that are greater than 35% for this biobrick. This also means that there is not of potential allergen status.

Tsinghua 2018's characterization

The Relation between LacI Concentration and IPTG Induction Efficiency

I Background

Tac promotor is a hybrid between trp and lacUV5 promoters. Tac promotor is a kind of strong E. coli promotors and is IPTG inducible. We design devices(K2558203/K2558204/K2558205) contain constitutively (BBa_J23100, Anderson strong/medium/weak promotor) expressed lacI protein and Ptac controlled sfGFP reporter, aiming to specify the relation between the level of lacI production and the quality of IPTG induced expression.

II Results

With high level of lacI expression (https://parts.igem.org/Part:BBa_K2558203), sfGFP fluorescence has almost no response to IPTG induction. Weak lacI expression (https://parts.igem.org/Part:BBa_K2558205) has the most significant IPTG induced sfGFP expression. With medium lacI concentration (https://parts.igem.org/Part:BBa_K2558204), the induction efficiency lies in between. Therefore, the result proves that high level of lacI expression severely decrease IPTG induction efficiency [1]. Furthermore, IPTG concentration can affect the regulation part performance. The figure shows that without IPTG the sfGFP florescence intensity will be at floor level. After IPTG addition, fluorescence signal immediately begins to climb, forming a peak at five hours after induction, then sfGFP florescence intensity will decrease and maintain at a lower level afterwards. IPTG concentration does not significantly affect the height of the peak or the expression level after the peak, but the peak width and expression stability of the system. Figures indicate that 5-10 mM IPTG has the most stable induction results.

III Experiment Design and Measurement Methods

In order to investigate how lacI dosage affect IPTG induction we used Anderson promotor J23100, J23110 and J23114 to design three constitutive lacI generator of different intensity. The three lacI generator were then ligated with Ptac controlled reporter sfGFP to make three IPTG induction devices (https://parts.igem.org/Part:BBa_K2558203, https://parts.igem.org/Part:BBa_K2558204, https://parts.igem.org/Part:BBa_K2558205). By measuring sfGFP fluorescence we tested how these devices react to IPTG.

Detailed measurement methods are as follows:

1. one Transform the plasmids into E. coli BL21(DE3) or DH5α.
2. two 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 5ml M9 medium with ampicillin at 100 ng/µl. Incubate for 6-8 h at 37℃ in a shaker.
3. three Measure OD600 of the culture medium with photometer. Dilute the culture medium until OD600 reaches 0.6.
4. four Add 100 µl bacteria culture medium into a sterile 96-well plate. Add IPTG to final concentrations of 0, 1, 5, 10, 20 mM. Fresh M9 medium serves as blank control. Positive control is colony constantly expressing sfGFP and negative control is colony without sfGFP expression. Place the 96-well plate into an automatic microplate reader. Incubate at 16℃ overnight and record the fluorometric value at 510 nm and OD600 for each well every 30 minutes.
5. five Each group is repeated for at least 3 times.

IV Reference

[1] Szabolcs Semsey, Sandeep Krishna. "The effect of LacI autoregulation on the performance of the lactose utilization system in Escherichia coli" Nucleic Acids Res 2013 Jul; 41(13): 6381–6390