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

In order to investigate how lacI dosage affects IPTG induction, we used Anderson promotor J23100, J23110 and J23114 to design three constitutive lacI generators of different intensities. The three lacI generators were then ligated with Ptac driven reporter sfGFP to make three IPTG induction devices (BBa_K2558203, BBa_K2558204, BBa_K2558205). By measuring sfGFP fluorescence we tested how these devices react to IPTG.

II Results

With high level of lacI expression (BBa_K2558203), sfGFP fluorescence had almost no response to IPTG induction. Weak lacI expression (BBa_K2558205) had the most significant IPTG induced sfGFP expression. With medium lacI expression level (BBa_K2558204), the induction efficiency lay in between. Therefore, the result proves that high level of lacI expression severely decreases IPTG induction efficiency [1]. Furthermore, IPTG concentration can affect the regulation part performance. The figure shows that without IPTG the sfGFP florescence intensity remained low. After IPTG addition, fluorescence signal immediately began to climb, forming a peak at five hours after induction, then sfGFP florescence intensity decreased and maintained at a lower level afterwards. IPTG concentration did not significantly affect the height of the peak or the expression level after the peak, but rather the peak width and expression stability of the system. Figures indicate that 5-10 mM IPTG had the most stable induction results.

Figure.1. The effect of varied lacI-LVA (BBa_C0011) promotor strength on IPTG induction of Tac promotor (BBa_K2558004). Relative fluorescent intensity is fluorescence per OD600 standardized with fluorescence per OD600 value of each test group at Time=0, IPTG=0. The promotors we used are from the Anderson collection: BBa_J23100 for strong lacI-LVA expression (pink), BBa_J23110 for medium expression (green), BBa_J23114 for weak expression (orange).

III Protocol

1. one Transform the plasmids into E. coli 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 should be 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