Part:BBa_K2612001
4-HT Intein in Kanamycin Resistance
This part consists of a split Kanamycin Resistance gene between the part BBa_J32009.
Characterization of insolubility of a small-molecule triggered intein splicing system in E. Coli
The goal of this year’s project was to create a biosensor that would luminesce in the presence of cortisol, giving our team the ability to quantify the hormone in saliva. The idea was ignited from an small-molecule triggered intein previously developed by Buskirk et al (2004). In this article, the authors inserted the ligand binding domain of the Estrogen Receptor into the RecA intein, disrupting its ability to perform auto-splicing, as found in nature. Splicing was only restored after the addition of the estrogen antagonist 4-hydroxytamoxifen (4-HT), and rounds of mutagenesis. The construct was then inserted into variety of split extein contexts, such as kanamycin resistance, and GFP, and found to splice together the split proteins halves and produce the mature protein, in a dose-responsive manner to 4-HT addition.
We sought to expand on the detection capabilities of this system, by creating a modular system, where both the ligand binding domain, and the extein could be replaced. The ultimate goal of the project was to create a biological sensor for the detection of cortisol. We would achieve this by swapping the estrogen receptor ligand binding domain for the glucocorticoid receptor, and replace the kanamycin resistance extein with the bright luciferase, NanoLuc.
Nuclear receptors such as estrogen and glucocorticoid share large homology in their DNA sequence, and 3D strucutre. The generic LBD of nuclear receptors is comprised of 12 a-helices and 4 small β-sheets that fold into a three-layer helical domain. Upon binding with an antagonist or agonist, the receptors undergo a conformational change. The conformational change is highlighted in the movement of helix 12. Our team modeld a variety of linkers for the glucocorticoid, which would connect the halves of the RecA intein upon cortisol binding We wanted linkers long enough to be able to wrap around the molecule upon conformational change and binding with the correct ligand, however the linkers needed to be rigid and short enough, not to induce auto-splicing without the need of ligand binding. The following linkers were designed based on the optimal angstrom distances and flexible design. Our dry lab team calculated that for a rigid linker, the shortest possible distance needed when the conformational change was initiated was 8 angstrom, and 20 angstrom for a more flexible linkers. We tried out three different linkers for our glucocorticoid construct, as we were unsure how rigid or flexible the linker needed to be to ensure splicing. Once the linkers were chosen, we performed molecular modelling to ensure that the two inteins halves would come into close enough contact to initiate splicing, and thus produce luminescence. The molecular dynamic simulations verified that the linkers: N-terminal: GPGSGS, C-terminal: GASGSG, were in fact long enough for the intein to come in contact, and they even formed a favourable interaction in just 10 nanosecond of simulation time. (see Molecular Dynamic Simulations her). Armed with knowledge gained in our dry lab, we set out to recreate the results by Buskirk et al. (2004), and develop new constructs based on the glucocorticoid receptor.
For proof of concept, we conducted experiments on these constructs in Eschiceria coli. Although the original paper utilized yeast, we set out to test the ability of the 4-HT dependent intein to splice in bacteria, in efforts to improve its characterization, and due to the relative ease of working with novel genetic constructs in yeast. We firstly constructed the Kanamycin-4HT-Intein, and Kanamycin-GR-Intein in the expression vector pET16b via gibson assembely with overlapping sequence, and then transformed DH5-alpha. We picked colonies, grew liquid cultures, mini-prepped, and performed diagnostic digests to confirm our insert was present.
We then transformed our construct into BL-21 DE3, a strain of E. coli that is important for protein expression, and induced expression with IPTG. To test intein splicing, we made agar plates containing either 1) Ampicillin, as a positive control, 2) Kanamycin, as a negative control 3) Kanamycin and 4-HT. We plated the BL21 DE3 and found that no bacterial colonies grew on the Kanamycin alone, or Kanamycin and 4-HT containing plates, but there was growth on the Ampicillin containing plates. If the iintein was in fact splicing caused by 4-HT, we would expect to see growth on the Kanamycin and 4-HT containing plate. Therefore, this demonstrated that the intein construct was not splicing and Kanamycin Resistance was not being produced.
To discern why the intein was not splicing we ran a SDS-page on induced BL21-DE3 expression. We grew 2 liquid cultures of BL21 DE3 containing the intein, induced expression of T7 RNA polymerase with IPTG, then incubated both for 4 hours at 37C. We then added 10μm 4-HT to one of the liquid cultures and incubated at 37C for an additional 1 hour to allow splicing of the intein to occur.
The team lysed the cells by addition of lysozyme and protease inhibitor containing lysis buffer, followed by sonication for 10 seconds. 10 seconds would provide sufficient enough time for splicing without destroying the cells, or having the liquid bacteria overheat. We then collected 1ml of the mixed fraction and stored at -20C. The remainder of the fraction were centrifuged at 8k RPM for 5 minutes and collected 1ml of the soluble fraction. The remainder of the soluble fraction was discarded, and the insoluble fraction was resuspended, and 1 ml was collected as the insoluble fraction.
We then ran a SDS-page to determine protein expression, and if splicing had occurred.
The SDS page informed us that the majority of our protein product was found in the pellet and not the supernatant. This indicated that our construct was not soluble, and would not be able to splice.
We subsequently found one paper that had previously utilized dose-dependent intein splicing in bacteria, however this paper further tagged intein constructs with a maltose-binding protein.[2] Therefore, this further demonstrates the 4-HT dependent intein to be insoluble in Bacteria, unless its solubility is drastically increased through the use of solubility tags such as Maltose-binding protein.
Our next step was to try and increase our protein’s solubility. Through a collaboration with the Stonybrook iGEM team, we decided to try directed evolution through error-prone PCR. We created primers for the insert and the backbone, and added MnCl¬2 to our PCR reaction to induce random mutagenesis. We then tried Gibson assembly and transformation of the mutagenized product to see if any insert or backbone had worked. If colonies grew on kanamycin and cortisol, we know that splicing worked. The colonies would then be sampled and plated on kanamycin, if they grew, then the intein was not selective for just ligand binding. Unfortunately, we saw no evidence of the error prone PCR being successful after numerous attempts.
In the future, our constructs solubility could be increased through error-prone PCR, with a careful selection process. As well, we discussed the possibility of adding a maltose binding domain to our protein and increasing its solubility. E. coli maltose binding protein (MBP), can be used to promote solubility of the protein that it is fused with.
Since the intein construct previously worked in yeast, we also believe our construct could be successfully constructed and spliced upon ligand binding in this organism.
Usage and Biology
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 690
Functional Parameters
| None |