Team:Tec-Monterrey/Parts

Overview

In designing the E. coding system, composite parts BBa_K2761009, BBa_K2761010, and BBa_K2761011 were built based on the work of Núñez et al. and Farzadfard et al. They include new basic parts for the iGEM Registry, as well as some improved parts from previous fellow iGEM Teams. Each new basic part is included and described separately from the corresponding new composite part in which it is used for the E. coding system.

Basic Parts

Retrotranscriptase EC86

Protein gene for the expression of retrotranscriptase protein EC86 from Escherichia coli including a His tag for its identification. The retrotranscriptase (RT) is a special protein capable of retrotranscript an RNA molecule into a DNA molecule. In the case of the EC86, it works along with a special msd-msr to form a Retron. Once transcribed, the msr-msd RNA folds into a secondary structure guided by the base pairing of the inverted repeats and the msr-msd sequence. The RT recognizes this secondary structure and uses a conserved guanosine residue in the msr as a priming site to reverse transcribe the msd sequence and produce a hybrid RNA-ssDNA molecule called msDNA (i.e., multicopy single-stranded DNA).

Wild Type Cas1 Protein

Protein gene for the expression of Cas1 from Escherichia coli in its wild type form. This protein is one of the two Cas proteins responsible for new DNA spacer acquisition in the bacteria's CRISPR locus (array). This action is done by a protein complex hexamere made up of 4 Cas1 dimers and 2 Cas2 dimers. It has been shown that these proteins are commonly conserved across the major types of CRISPR-Cas systems. To properly use Cas1 protein it should be expressed along with Cas2 protein in the system design for spacer acquisition. The Cas1 protein gene contained in this part expresses the protein without any tag for further identification.

Wild Type Cas2 Protein

Protein gene for the expression of Cas2 from Escherichia coli in its wild type form. The basic part includes Cas2 protein from Escherichia coli. This protein is one of the two Cas proteins responsible for new DNA spacer acquisition in the bacteria's CRISPR locus (array). This action is done by a protein complex hexamere made up of 4 Cas1 dimers and 2 Cas2 dimers. It has been shown that these proteins are commonly conserved across the major types of CRISPR-Cas systems. To properly use Cas2 protein it should be expressed along with Cas1 protein in the system design for spacer acquisition. The Cas2 protein gene contained in this part expresses the protein without any tag for further identification

Cas1 Protein with Flag

Protein coding gene for the expression of Cas1 from Escherichia coli including a FLAG tag for its identification and purification. This protein is one of the two Cas proteins responsible for new DNA spacer acquisition in the bacteria's CRISPR locus. This action is done by a protein complex hexamere made up of 4 Cas1 proteins and 2 Cas2 proteins. It has been shown that these proteins are commonly conserved across the major types of CRISPR-Cas systems.

Cas2 Protein with HA

Protein coding gene for the expression of Cas2 from Escherichia coli including a HA tag for its identification and purification. This protein is one of the two Cas proteins responsible for new DNA spacer acquisition in the bacteria's CRISPR locus. This action is done by a protein complex hexamere made up of 4 Cas1 proteins and 2 Cas2 proteins. It has been shown that these proteins are commonly conserved across the major types of CRISPR-Cas systems.

NsrR

This basic part consists of an optimized NsrR protein gene, which acts as a repressor of PyeaR promotor and is inhibited by the nitrate ion. E. coli natively produces a certain amount of NsrR. As such, the overexpression of NsrR can reduce any basal expression of a PyeaR construct that may not be silenced by native NsrR. With a PyeaR reporter construct, NsrR can be used to determine the presence of nitrate ion.

RBS

RBS sequence used for the expression of Cas1 and Cas2 proteins.

msr-msd Scaffold

Sequence for msr-msd transcript from EC86 retron, with specific PAM sequence for Cas1-Cas2 recognition. The msd-msr is transcribed as a single RNA sequence, and interacts with the Retrotranscriptase (RT) EC86 protein to form an RNA-DNA chimera. The msr serves as a primer for the RT to start the retrotranscription, and the msd is the part of the transcript that is converted back into DNA. The msd is composed of a conserved part and a variable part. The conserved part is needed for retrotranscription and retron stability, while the variable part may be changed for any sequence of interest. In this part, the variable part of the msd contains an improved sequence that have high integration rates in the CRISPR array by adaptation proteins Cas1 and Cas2.

Composite Parts

Cas1-Cas2 proteins

The composite parts BBa_K2761007 and BBa_K2761009 hold an IPTG-inducible promoter regulating the expression of each Cas1 and Cas2 proteins. The first part produces each protein with a FLAG and HA tags, respectively, the second one produces them as wild type proteins. The expression of the proteins was characterized, quantifying them with a gradient of inductor concentration and under different induction times. The effect of different induction times is displayed on Figure ***: Time does things***. The effect of inductor concentration can be seen on Figure ***: Concentration does things***.

RT & msd-msr

The composite part BBa_K2761010 holds an IPTG-inducible promoter regulating the expression of a Retrotranscriptase and the msr-msd transcript. The expressed RT comes with a Histidine tag. The RT cassette for the new parts were improved from Part BBa_K1681000 by iGEM Team UIUC Illinois. However, the new parts are designed with a different msr-msd transcribed under a separate promoter cassette.
The expression of the protein was characterized, quantifying it with a gradient of inductor concentration and under different induction times. The effect of different induction times is displayed on Figure ***: Time does things. The effect of inductor concentration can be seen on Figure ***: Concentration does things.

Nitrate inducible promoter

As part of the environmental perspective of our project, a promoter inducible by the nitrate ion was designed, based on part BBa_K381001 by iGEM Team BCCS-Bristol. The original composite part consists of a promoter silenced by a protein NsrR, which is natively produced by Escherechia coli, and the cassette for expression of a GFP. NsrR protein is inhibited by the nitrate ion, so that in its presence the cassette expresses the GFP and fluorescence is observed. This fluorescence can be calibrated and nitrate concentration in a solution can be determined from the bacteria’s fluorescence.
For our system, we introduced a constitutive promotor expressing NsrR, so that the other promotor is better silenced, with the intent of giving the fluorescence response greater sensibility to nitrate ion.
The new part is ligated to BBa_K381001, to make a new composite part, BBa_K2761011, that is more sensitive to the analyte, nitrate ion.
Under the same mechanism of inhibiting the repressor protein NsrR by action of nitrate ion, the E. coding system can be adapted so that the production of Cas proteins and a target DNA sequence occurs only on the presence of this analyte.

Composite part BBa_K2761011 characterization
This nitrate sensitive system was tested for fluorescence, constructing a calibration curve of Fluorescence versus Nitrate concentration. To test the system’s reliability, bacteria were induced with a known concentration of nitrate ion, their fluorescence was measured and interpolated in the calibration curve. The measured value of nitrate concentration was compared with the known value to evaluate the system’s error for measuring nitrate concentration.
The calibration curve was constructed with the concentrations and induction times as displayed in Table ***.
[Tabla parámetros]

The constructed calibration resulted in an Fluorescence versus Nitrate concentration equation: y = mx + b. Where y is Fluorescence divided by Absorbance at 600 nm, x is Nitrate concentration in mM, m the curve’s slope, and b the intersection with the y-axis. The linear regression results with an R^^^2 of ****.
[Gráfica]

Testing the system with a known concentration of nitrate ion, the concentration determined by its fluorescence interpolating in the calibration curve. The result yields a relative error of ***%.

On the other hand, as seen in Figure ***, basal expression of the GFP cassette was successfully further silenced the overexpression of NsrR of the second cassette. Fluorescence follows a linear relationship to nitrate concentration from minimum to more significant values, enabling a wider range of sensibility for nitrate determination, leaving an insignificant amount of basal fluorescence from the cassette leaking.
[Gráfica]

This enables this nitrate-sensitive system to be implemented in the E. coding system without false positives, from insertions happening by action of any basal expression of the system’s proteins. With the E. coding system, the nitrate ion can be detected and potentially quantified over long periods of time directly on-site.

Parts Collection

Team:Tec-Monterrey/Parts

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