Team:Hong Kong HKUST/riboregulator/regulatory RNAs catalog

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Catalog for regulatory RNAs

(The page was created as part of iGEM 2014 HKUST team's effort in "Project Riboregulator" to catalog existing regulatory RNAs. Over the years, the number of regulatory RNAs in Part Registry has steadily increased over time and many has been made available to end users. Based on different mode actions and natures of regulatory RNAs, they can be grouped into different categories. However, the Part Registry currently does not have a catalog page, categorizing methods or guidelines to organize and curate existing regulatory RNAs. Some of them are grouped under type "RNA", while others are not. This is not useful for looking up and utilizing them.

We would like to solve this problem by designing a list of category tags as well as a guideline, so that automated display of regulatory RNAs by the <parttable> function can be facilitated. By doing so, we hope that we can assist other users to find and use those parts efficiently.

This page was written in compliance with Part Registry's format for general Catalog Pages. Currently, the information is uploaded manually because we have yet to submit our suggestions to iGEM HQ. Upon approval, we will tag existing regulatory RNAs and complete the page. Being part of the cross-cohort "Project Riboregulator", the page is far from complete and is expected to take shape by Spring 2015. We welcome and encourage constant update and adoption of this page in the future.)

Guideline for adding Categories

Some guidelines for categorizing RNA devices under this scheme:

  1. Confirm it is non-coding RNA devices
  2. Give category for “level of regulation”
  3. List the natures of RNA devices currently under exam (the RNA devices)
  4. Compare with the properties of different types of RNA devices with the RNA devices
  5. Assign categories about “nature of part” to the RNA devices currently under exam only if the RNA device fulfils all the requirements listed for any type of RNA devices
    1. Assign “//RNA/ncRNA/others” if no matches
    2. Assign “//RNA/ncRNA/target_sequence” if it is target sequence of other ncRNA devices; Be sure also assign corresponding categories of ncRNA devices. For example, target sequence of sgRNA should be assigned for :
      • //RNA/ncRNA/target_sequence
      • //RNA/ncRNA/sgRNA
  6. Assign categories of “function of part”. Note that it may not be limited to the two function listed above. Check the existing categories to assign appropriate categories.

Riboregulator

Riboregulators regulate translation by having two elements, a cis-repressive sequence upstream of RBS in mRNA, and a non-coding RNA device, called trans-activating RNA. The cis-repressive sequence will binds to the 5'UTR, including the RBS by Watson-Crick base pairing, the sequestration of RBS represses translation. While trans-activating RNA will form complementary bases to cis-repressive sequence and exposing RBS for ribosomal binding and allow translation.

Proposed Categories: /RNA/non_coding/post_transcriptional/Riboregulator

Designer Part Number Description
Delft 2009 BBa_K175029 Weak lock
Delft 2009 BBa_K175030 Key for lcok of weak RBS
Delft 2009 BBa_K175030 Medium lock
Delft 2009 BBa_K175030 Key for Medium lock
Delft 2009 BBa_K175034 (Constitutive expression of GFP with weak RBS lock and inducible production of key for the lock Composite of K175029 + K175030
Delft 2009 BBa_K175034 Constitutive expression of GFP with medium RBS lock and inducible production of key for the lock Composite of K175031 + K175032
Caltech 2007 BBa_I759015 cis3-repressed, tet-regulated YFP
Caltech 2007 BBa_I759016 cis4-repressed, tet-regulated YFP
Caltech 2007 BBa_I759020 cis8-repressed, tet-regulated YFP
Caltech 2007 BBa_I759027 cis3-repressed, tet-regulated Q
Caltech 2007 BBa_I759028 cis4-repressed, tet-regulated Q
Caltech 2007 BBa_I759014 (cis2-repressed, tet-regulated YFP
Caltech 2007 BBa_I759017 cis5-repressed, tet-regulated YFP
Caltech 2007 BBa_I759018 cis6-repressed, tet-regulated YFP
Caltech 2007 BBa_I759019 cis7-repressed, tet-regulated YFP
Caltech 2007 BBa_I759013 cis1-repressed, tet-regulated YFP
Caltech 2007 BBa_I759032 Ptet_cis1_YFP
Caltech 2007 BBa_I759034 Ptet_cis2_YFP
Caltech 2007 BBa_I759036 Ptet_cis3_YFP
Caltech 2007 BBa_I759038 Ptet_cis4_YFP
Caltech 2007 BBa_I759040 Ptet_cis5_YFP
Caltech 2007 BBa_I759042 Ptet_cis6_YFP
Caltech 2007 BBa_I759044 Ptet_cis7_YFP
Caltech 2007 BBa_I759046 Ptet_cis8_YFP
Caltech 2007 BBa_I759023 pBAD-trans2
Caltech 2007 BBa_I759022 pBAD-trans1
Caltech 2007 BBa_I759024 pBAD-trans3
Caltech 2007 BBa_I759025 pBAD-trans4
Caltech 2007 BBa_I759026 pBAD-trans5
Peking 2007 BBa_I714070 R0040-J23078-pTet-Lock3
Peking 2007 BBa_I714080 [R0040][J23078][E0040][B0015]
Peking 2007 BBa_I714081 R0040-J01010-E0040-B0015
Peking 2007 BBa_I714037 R751+ C600 E.coli cells with traI-R751 knockout
Peking 2007 BBa_I714074 R0010-J23066-pLac-Key3-DblTerm Uses Lock and Key 3 from berkeley
K.U. Leuven 2008 BBa_K145215 FILTER Key (TetR promoter + key)
K.U. Leuven 2008 BBa_K145216 FILTER T7 RNA pol Lock from berkeley
K.U. Leuven 2008 BBa_K145217 FILTER Complete The two previous together
K.U. Leuven 2008 BBa_K145220 INVERTED TIMER
K.U. Leuven 2008 BBa_K145225 RESET lactonase
K.U. Leuven 2008 BBa_K145300 Lactonase controlled by key/lock
K.U. Leuven 2008 BBa_K145301 lacI controlled by key/lock
K.U. Leuven 2008 BBa_K145302 luxI generator controlled by key/lock
K.U. Leuven 2008 BBa_K145303 GFP generator controlled by key/lock
K.U. Leuven 2008 BBa_K145003 T7 PoPS -> RiboKey 3d
K.U. Leuven 2008 BBa_K145004 T7 PoPS + RiboLock |> LuxI
K.U. Leuven 2008 BBa_K145005 T7 PoPS + PR -> cI
K.U. Leuven 2008 BBa_K145216 FILTER T7 RNA pol
K.U. Leuven 2008 BBa_K145251 OLD RESET lactonase
K.U. Leuven 2008 BBa_K145253 OLD INVERTIMER Part 1
K.U. Leuven 2008 BBa_K145255 NEW INVERTIMER part 1
K.U. Leuven 2008 BBa_K145264 test FILTER (new)
K.U. Leuven 2008 BBa_K145265 test FILTER (old)
K.U. Leuven 2008 BBa_K145271 GFP regulated by AND-gate
K.U. Leuven 2008 BBa_K145272 GFP regulated by AND-gate
K.U. Leuven 2008 BBa_K145275 T7 polymerase generator under TetR repressible promoter (filter)
K.U. Leuven 2008 BBa_K145276 T7 polymerase generator under TetR repressible promoter
K.U. Leuven 2008 BBa_K145277 T7 DNA polymerase regulated by lock
K.U. Leuven 2008 BBa_K145278 T7 DNA polymerase regulated by [lock3d]
K.U. Leuven 2009 BBa_K238004 Vanillin synthesis
K.U. Leuven 2009 BBa_K238006 Short version of vanillin synthesis
K.U. Leuven 2009 BBa_K238012 short version II of vanillin synthesis
Groningen 2011 BBa_K607005 short version II of vanillin synthesis
Groningen 2011 BBa_K607000 PhybB_taRNA
VictoriaBC 2009 BBa_K235010 [K145303] (ribokey-controlled GFP generator)
VictoriaBC 2009 BBa_K235000 [R0010][J23066] (pLac+ribokey+stop)
VictoriaBC 2009 BBa_K235001 [J23102][J23066] (constitutive promoter+ribokey+stop)
VictoriaBC 2009 BBa_K235009 [J23102][J23032] (constitutive promoter+ribolocked RBS)
VictoriaBC 2009 BBa_K235011 [K235009][K235005] (ribokey-controlled mCherry generator)
VictoriaBC 2009 BBa_K235013 [K145303][K235000] (ribokey-mediated pLac-controlled GFP reporter)
VictoriaBC 2009 BBa_K235014 [K145303][K235001] (ribokey-mediated GFP generator)
VictoriaBC 2009 BBa_K235016 [I0500][J23032] (pAra+ribolocked RBS)
VictoriaBC 2009 BBa_K235019 [K235016][K235003] (ribokey-mediated pAra-controlled lambda repressor generator)
VictoriaBC 2009 BBa_K235021 [K235009][K235003] (ribokey-mediated lambda repressor generator)
VictoriaBC 2009 BBa_K235022 [K235018][K235019] (mCherry generator, pAra-controlled ribokey-mediated signal inversion)
VictoriaBC 2009 BBa_K235024 [K235018][K235021] (mCherry generator, ribokey-mediated signal inversion)
VictoriaBC 2009 BBa_K235025 [K235022][K235000] (NAND gate, pAra and pLac input signal control, mCherry output signal)
VictoriaBC 2009 BBa_K235026 [K235022][K235001] (NAND gate control test, pLac positive control)
VictoriaBC 2009 BBa_K235027 [K235024][K235000] (NAND gate control test, arabinose positive control)
VictoriaBC 2009 BBa_K235028 [K235024][K235001] (NAND gate control test, positive control)
Melborne2008 BBa_K085000 (lacI)promoter->key3c
Melborne2008 BBa_K085002 pTet->lock3d->GFP
Calgary 2007 BBa_I737003 OmpF controlled RNA Key
Calgary 2007 BBa_I737006 Temperature induced repression/activation of an RNA key
Calgary 2007 BBa_I737005 AHL and RNA lock controlled AraC

RNA Aptamer

A RNA apatamer is any RNA molecule that can fold into a tertiary confirmation that binds with strong affinity and high specificity to small molecules through non-Watson-Crick base pairing.

Designer Part Number Description

Riboswitch

A riboswitch is a segment on mRNA that has the ability to detect small molecules or temperature, and regulates gene expression in an on or off manner. Riboswitches usually contain sensor domain for binding of small molecules and a regulatory domain for gene regulation. Riboswitches are therefore also aptamers in nature. Upon binding of a suitable ligand in the sensor domain, riboswitches undergo confirmation changes that can lead to different outcomes like translation inhibition or mRNA degradation.

Designer Part Number Description

RNA-IN-RNA-OUT

RNA-OUT is a small non-coding RNA that works at RNA level . RNA-OUT will bind to 5'UTR, which include RBS, of mRNA and prevents ribosome from binding to mRNA to inhibit translation of downstream gene. RNA-IN is also a non-coding RNA that is antisense to RNA-OUT and the binding of RNA-IN and RNA-OUT will prevent RNA-OUT from binding to mRNA, thus allowing ribosome to bind to mRNA and initiate translation.

Designer Part Number Description

Antisense RNAs (asRNAs)

asRNAs are single stranded RNAs that usually form base pair extensively with the target sense RNA / DNA. RNAs belonging to this class are capable of blocking translation, interfering with transcription, or modulating RNA stability. A review by Thomason and Storz provided detailed descriptions to their properties and functions.

Reference:

Thomason MK, Storz G.,Bacterial antisense RNAs: how many are there, and what are they doing?Annual Review of Genetics Vol 44:167±188(2010)

Designer Part Number Description

Small interfering RNAs (siRNAs)

siRNAs work in the RNA interference (RNAi) pathway. siRNAs are usually produced by "dicing" exogenous, long double stranded RNA into 21-nucleotides small fragments. The siRNAs will then bind to Argonaute in RNA-induced silencing complex (RISC) and one strand is discarded. The remaining strand then guides the RISC to RNA targets with complementary base pairs and in most cases degrades the latter, result in inhibition of gene expression.

Designer Part Number Description

microRNAs (miRNAs)

miRNAs, like siRNA, work through the RNA interference (RNAi) pathway They are usually 19-25nt single stranded RNA that have an endogenous origin. They started as hairpin transcripts and processed by Drosha in non-random manner and are then processed by Dicer. Following binding to RISC, they regulate normal biological processes by degrading or inhibiting mRNAs that it has complementarity to.

Designer Part Number Description

Single guiding RNA (sgRNAs)

sgRNAs work in the CRISPR/Cas system. They are constructed by fusing functional domains of CRISPR RNA (crRNA) and trans-acting crRNA (taRNA) together through RNA linkers. They associate with Cas9 proteiens or their derivatives and guide them to DNA with complementarity with the targeting sequence of crRNA.

Designer Part Number Description

Ribozyme

A ribozyme is a RNA molecule with intrinsic catalytic activity, that can catalyse conversion of substrate into a product.

Designer Part Number Description

Aptazymes

RNA aptazymes, as the name suggests, are RNAs that carry properties from both aptamers and ribozymes. They are capable of sensing small molecules. Upon activation by a ligand, they can trigger ribozyme-mediated cleavage.

Designer Part Number Description

Structural Scaffolds

These ncRNAs have multiple interaction domains concatenated into a single molecule so as to facilitate co-localization of functional modules.

Designer Part Number Description

pT181-RNAI

The pT181-RNAI is a special class of regulatory RNAs derived from elements in Staphylococcus aureus pathogenicity plasmid pT181. A specific 5’ UTR region would normally form an anti-termination loop. Upon interacting with a pT181-RNAI, a premature terminator loop 5’ to the CDS will form and result in early termination before the mRNA can be completely transcribed.

Designer Part Number Description

Stability Control Elements

This class of ncRNAs can help to stabilize or destabilize a RNA molecule, typically by giving rise to hairpin structures that block or facilitate access of ribonucleases to the RNA itself.

Designer Part Number Description

Complex

This category contains ncRNA with dual or more functions resulting from combining 2 or multiple natures / functions of existing ncRNAs.

Designer Part Number Description

Targeted Sequence

This category deals with segments of ncRNAs that serve as recognizable targets by other ncRNAs.

Designer Part Number Description

Others

ncRNAs not belonging to the any of the above categories are listed here.

Designer Part Number Description

Differentiation

siRNA VS miRNA

siRNA and miRNA are two very similar RNA devices. Both of them will be processed by Dicer and both of them will from a RISC complex to carry out their function. However there are substantial differences between the two.

First, siRNA are 20 to 25 nucleotides long; while miRNAs are 19-25 nucleotides long.

Second, siRNA usually fully complement with the target mRNA; while miRNA can be partially complement with target mRNA.As a result, siRNA usually target few mRNA while miRNA can target 250-500 different mRNAs. Last but not least, siRNAs usually stem from exogenous DNA; while miRNA usually stem from endogenous DNA.

siRNA / miRNA VS asRNA

asRNAs refers to single stranded RNAs that are complementary to mRNA. Although no specific length requirement is imposed on asRNAs, asRNAs usually refers to RNA with longer length by historical reason. Whereas siRNA refers to short double stranded RNAs that are 20-25 nt long; miRNA usually refers to single stranded RNA that are 19-25 nt long. asRNA form duplex with mRNA, which will blocks the access of ribosome to mRNA, also the duplex may be degraded by ribonuclease exist in the cell. Either functions of siRNA and miRNA depends on RISC.

Structural scaffold VS Stability control elements

Structural scaffold refers to the folding of RNA, which has the ability to recruit various molecules, mostly proteins. Scaffold may stabilize RNA devices and therefore a scaffold can also be stability control elements. While stability control elements refers to any RNA devices that contribute to the stability of RNA devices. It is not limited to RNA scaffold. One example is poly A tail elements for mRNA, it contributes to the stability of mRNA, however it is not a loop.

Case Study

Case one: Spinach Aptamer

Type of Category THE PROPOSED CATEGORIES
FUNCTION OF PART//RNA/ncRNA/function/reporter
LEVEL OF CONTROLNone
NATURE OF PART//RNA/ncRNA/aptamer
Chassis//Chassis/prokaryote/ecoli
//Chassis/eukaryote/human
Justification

FUNCTION OF PART : //RNA/ncRNA/function/reporter
Spinach RNA aptamer binds with fluorophores mimics the GFP, and RNA fluorophore complex emit green fluorescence upon exposure of UV light.

LEVEL OF REGULATION: NONE
Since this RNA devices is not involved in gene regulation, it is not assigned with any category related to level of regulation.

CHASSIS: //Chassis/prokaryote/ecoli & //Chassis/eukaryote/human
There are evident that part are functional in the two chassis.

Case two: RNA_OUT

Type of Category THE PROPOSED CATEGORIES
FUNCTION OF PART//RNA/ncRNA/function/regulation
LEVEL OF REGULATION//RNA/ncRNA/function/regulation/RNA_level
NATURE OF PART//RNA/ncRNA/RNA_OUT_type/ RNA_OUT
//RNA/ncRNA/asRNA
Chassis//Chassis/prokaryote/ecoli
Justification

FUNCTION OF PART : //RNA/ncRNA/function/regulation
RNA_OUT binds to upstream of CDS of mRNA preventing ribosomal binding, thus down-regulate gene expression.

LEVEL OF REGULATION://RNA/ncRNA/function/regulation/RNA_level
RNA_OUT regulate gene expression by interacting with mRNA. Since it’s target is a RNA and it is exerting gene regulation.

NATURE OF PART: //RNA/ncRNA/RNA_OUT_type/ RNA_OUT
This device fit all the requirements for RNA_OUT as mention.

  1. is asRNA
  2. contains stem and loop where loop can interact with RNA_IN
  3. base pair with RNA_IN will complement to 5’ of mRNA and block access of RBS from ribosome
  4. active in trans

NATURE OF PART: //RNA/ncRNA/asRNA
This device fit all the requirements for RNA_OUT as mention. This RNA device will form complementary base pairs with mRNA, also it is a single stranded RNA. Since it fulfil all requirement of antisense RNA, It is assigned to this category.

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