Team:Tec-Monterrey/Parts
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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 which are used for the E. coding system.
Basic Parts
Retrotranscriptase EC86: BBa_K2761000
Gene sequence for the expression of retrotranscriptase protein EC86 from Escherichia coli including a histidine tag for its identification and purification. Main component of the SCRIBE (i.e, Synthetic Cellular Recorders Integration Biological Events). The retrotranscriptase (RT) is a special protein capable of retrotranscribing a RNA molecule into a DNA molecule. In the case of the EC86, it acts on a special sequence of RNA (msr-msd), a non-coding RNA. Once transcribed, the msr-msd sequence folds into a secondary structure guided by the base pairing of the inverted repeats that flank this 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).The msDNA contains the synthetic message that would be inserted in the bacteria genome for the memory acquisition.
Wild Type Cas1 Protein: BBa_K2761001
Gene sequence 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 and a PAM sequence found in the msDNA in the system design for spacer acquisition. The Cas1 protein gene contained in this part expresses the protein without any tag for further identification.
Cas1 Protein with Flag: BBa_K2761003
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. To properly use Cas1 protein it should be expressed along with Cas2 protein and a PAM sequence found in the msDNA in the system design for spacer acquisition. The Cas1 protein gene contained in this part expresses the protein with a tag sequence (FLAG) for further identification and purification.
Cas2 Protein with HA: BBa_K2761004
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.To properly use Cas2 protein it should be expressed along with Cas1 protein and a PAM sequence found in the msDNA in the system design for spacer acquisition. The Cas2 protein gene contained in this part expresses the protein with a tag (HA) for further identification and purification.
NsrR: BBa_K2761005
This basic part consists of an optimized NsrR protein gene, which acts as a repressor of PyeaR promoter 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 GFP expressed by the PyeaR construct BBa_K381001 that may not be silenced by native NsrR. With a PyeaR reporter construct, the NsrR can be used to determine the presence of nitrate ion without any leaking.
RBS for Cas1 and Cas2 proteins: BBa_K2761006
RBS sequence used for the expression of Cas1 and Cas2 proteins.
msr-msd Scaffold: BBa_K2761008
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
New Parts: Cas1-Cas2 proteins: BBa_K2761007, BBa_K2761009
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. These are new characterized parts for Silver requirements. The characterization of the part can be found in the Demonstrate segment.
Improved Part: RT & msd-msr: BBa_K2761010
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. This characterized part is an improvement of a previous part for Gold requirements. The characterization of this part can be found in the Demonstrate segment.
Improved Part:Nitrate inducible promoter: BBa_K2761011
The composite part BBa_K2761011 contains the NsrR gen regulated by a constitutive promoter. This segment was ligated to BBa_K381001 which contains a Pyear promoter and a GFP reporter gen. Pyear is inhibit by the presence of NsrR which is natively produced by the bacteria. Under this condition no fluorescence is expected. Still is reported that under this condition, there is leaking activity that produces GFP. An over expression of the NsrR protein was proposed to overcome this problem. The presence of nitrate inhibits the NsrR protein from interfering with the promoter. This improvement intents to give the fluorescence response greater sensibility to nitrate ion. This characterized part is an improvement of a previous part for Gold requirements. The characterization of this part can be found in the Demonstrate segment.
Parts Collection
E-coding design a set of composite parts that enable the recording of stimuli from environment and culture conditions in the bacterial genome. The SCRIBE system together with the CAS1-CAS2 complex enables the integration of any designed sequence in the CRISPR-array. The project expects that the following collection of parts can be used in any type of bacteria for molecular recording of events. In this case, all experimentation was base in strain .BL21 (DE3) Further experimentation in other types of strain is encouraged.
- Cas1-Cas2 cassette with wild type proteins for integration in the bacteria CRISPR array
- Cas1-Cas2 cassette with tag proteins, Flag and HA respectively, for rapid identification of protein production and activity of integration.
- RT-His cassette for the production of a retrotranscriptase with a histidine tag. Cassette involves for the production of the desired sequence for integration of the genome and analog for the stimulus in measurement.
- NsrR cassette for the production of NsrR protein to reduce leaking activity in the Pyear promoter that leads to GFP production. This cassette is used primarily to sense the presence of nitrates in the environment.