Part:BBa_K2621000

CAT-Seq Esterase

CAT-Seq Esterase is a hydrolase used in Catalytic Activity Sequencing system to catalyse a reaction wherein a N4-benzoyl-2'-deoxycytidine triphosphate (Substrate Nucleotide) is converted into a 2'-deoxycytidine triphosphate (Product Nucleotide).

It is the main component of Catalytic Activity Sequencing (CAT-Seq) method. CAT-Seq is a method for high-throughput catalytic biomolecule and genetic regulatory part activity-sequence relationship assessment toolkit.

CAT-Seq Esterase can be used to analyse genetic transcriptional and translational regulatory part activities and their cross-interactions.

Also during the development of the CAT-Seq (Vilnius-Lithuania Overgraduate 2018), together with its in-silico derived mutants, this Esterase was used to assess the accuracy and precision of the system.

See how this part is used in the CAT-Seq by pressing here!

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 671
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 487
  
• 1000
  COMPATIBLE WITH RFC[1000]

Introduction

Biology

Description of the CAT-Seq esterase

Figure 1. Main principles of ColE1 plasmid family replication

CAT-Seq Esterase is a hydrolase used in Catalytic Activity Sequencing system to catalyse a reaction wherein a N4-benzoyl-2'-deoxycytidine triphosphate (Substrate Nucleotide) is converted into a 2'-deoxycytidine triphosphate (Product Nucleotide).

It is the main component of Catalytic Activity Sequencing (CAT-Seq) method. CAT-Seq is a method for high-throughput catalytic biomolecule and genetic regulatory part activity-sequence relationship assessment toolkit.

CAT-Seq Esterase can be used to analyse genetic transcriptional and translational regulatory part activities and their cross-interactions.

Also during the development of the CAT-Seq (Vilnius-Lithuania Overgraduate 2018), together with its in-silico derived mutants, this Esterase was used to assess the accuracy and precision of the system.

In-Silico design of the CAT-Seq esterase mutants

CAT-Seq Esterase is a hydrolase used in Catalytic Activity Sequencing system to catalyse a reaction wherein a N4-benzoyl-2'-deoxycytidine triphosphate (Substrate Nucleotide) is converted into a 2'-deoxycytidine triphosphate (Product Nucleotide).

ColE1-type plasmid replication begins with the synthesis of plasmid encoded RNA II (also called primer transcript) by RNA polymerase which initiates transcription at a site 555bp upstream of origin of replication. The RNA transcript forms a RNA - DNA hybrid with template DNA near the origin of replication. Hybridized RNA is then cleaved at the replication origin by RNAse H and serves as a primer for DNA synthesis by DNA polymerase I (Figure 1. A).

The interaction between RNA I and RNA II can be amplified by Rop protein, see part:BBa_K2259010.

Usage with CAT-Seq (Catalytic Activity Sequencing)

About CAT-Seq

Figure 1. Abstract scheme of the Catalytic Activity Sequencing

SynORI is a framework for multi-plasmid systems created by Vilnius-Lithuania 2017 which enables quick and easy workflow with multiple plasmids, while also allowing to freely pick and modulate copy number for every unique plasmid group! Read more about [http://2017.igem.org/Team:Vilnius-Lithuania SynORI here]!

Determining the accuracy of CAT-Seq

Trying to build a CAT-Seq pipeline in your own laboratory will require the CAT-Seq esterase in order to troubleshoot the system and assess the measurement accuracy and precision. In other words, the esterase can be used to calibrate the CAT-Seq.

Together with the Esterase, its substrate attached to a nucleotide is required (substrate nucleotide). In the standard case, the Substrate Nucleotide is N4-benzoyl-2'-deoxycytidine triphosphate. If the Esterase catalyzes the removal of the substrate from the nucleotide, it becomes the Product Nucleotide - 2'-deoxycytidine triphosphate.

Genetic Regulatory Part activity and cross-interaction assessment

It immediately becomes clear that in order to control the copy number of a plasmid one could simply change RNA I promoter. But, as RNA I and RNA II are two antisense molecules, changes made to the sequence will affect both of

Example for RBS activity strength determination

As RNA I and RNA II interact mainly with the three stem loops that form kissing complexes, we have decided to use this fact to our advantage in order to engineer different plasmid groups by adding unique, group-specific sequences to RNA I and RNA II stem loops.

For example if there are two plasmid groups in a cell - A and B - RNA II of A group
would only interact with RNA I A, and not RNA I B.

The inactivation and transfer of RNA I gene away from RNA II allow us to use different sequences for RNA I and RNA II molecules that are not necessarily ideal complements of each other.

Example for Toehold cross-interaction determination

In order to flexibly control the synthesis of RNA I, the RNA I gene first needed to be inactivated in the ColE1 origin of replication. That, however, was not a trivial task, because by changing RNA I promoter sequence, one also changes the RNA II secondary structure, which is crucial for plasmid replication initiation (Find how this problem was solved at [http://2017.igem.org/Team:Vilnius-Lithuania team Vilnius-Lithuania wiki]). This is the main reason why, in the SynORI framework, the wildtype ColE1 ORI is split into two different parts - RNR I and RNA II .

Characterization of the CAT-Seq Esterase (Vilnius-Lithuania Overgraduate 2018)

Esterase and its mutant activity measurement

Challenges of inactivating RNA I in wild type origin of replication were discussed earlier

After the construction of selected ColE1 mutants with inactivated RNA I promoter we have tested whether it was successful. It is difficult to distinguish when the promoter is fully disabled because first, there is no literature data describing replicons that are not negatively regulated at least to some extent, and second - plasmid systems hardly reach the equilibrium without negative control therefore every copy number calculation varies greatly. This is why we decided not to check for the highest copy number mutant, but rather to insert a wild type RNA I with its wild type promoter. By doing that we could see which replicons were most precisely mutated.

ORI 2 mutant seemed like a perfect candidate. Its copy number increased from wild type 37 copies to 1128 ± 315 copies in ORI2. In addition, when RNA I gene was placed next to it, the copy number of the constructed plasmid fell to wild type levels. After these results we have decided to use this ORI 2 mutant as a core for our framework. We simply called it RNA II (part:BBa_K2259000)

In silico produced mutant library based on CAT-Seq esterase enzyme

Once the RNA I promoter was disabled in the ColE1 origin of replication, it could be moved to a different plasmid location and used as a separate unit. We have discovered the sequence of wild type RNA I promoter by using PromoterHunter and removed it, thus creating a wild type RNA I gene part:BBa_K2259005. First, series of Anderson promoters were cloned next to the RNA I gene (part:BBa_K2259021 (0.15 Anderson), part:BBa_K2259023 (0.36 Anderson), part:BBa_K2259027 (0.86 Anderson), part:BBa_K2259028 (1.0 Anderson)) and then placed next to RNA II (part:BBa_K2259067 (0.15 Anderson), part:BBa_K2259068 (0.36 Anderson), part:BBa_K2259069 (0.86 Anderson), part:BBa_K22590671 (1.0 Anderson)).

Ribosome binding sites

When different groups of SynORI system were created, the abilty of corresponding RNA I to inhibit the replication of RNA II were measured by calculating the plasmid copy number with and without RNA I in the system

Toehold regulatory sequence library

As can be seen in Figure 7, RNA I introduction into the system has a significant effect on the plasmid copy number of the specific group, thus we can conclude that RNA I works on corresponding RNA II.