Team:USP SaoCarlos-Brazil/Basic Part

IARA

BASIC PARTS

TABLE OF PARTS

OUR PARTS

PART NAME

PART NUMBER

FUNCTION

MerR + Bidirecional promoter BBa_K3280005 MerR regulator protein
tDGC + rbs BBa_K3280000 Biofilm formation
MBP + linker BBa_K3280006 Metal binding peptide
Wspr BBa_K3280001 Biofilm formation

PREEXISTING PARTS

PART NAME

PART NUMBER

FUNCTION

YdeH BBa_K1019004 Biofilm formation
YddV BBa_K748003 Biofilm formation
TolC BBa_K554009 Part of protein secretion system
HlyB BBa_K554007 Part of protein secretion system
HlyA BBa_K554002 Signal peptide -Part of protein secretion
HlyD BBa_K554008 Part of protein secretion

BIOFILM FORMATION

YdeH

YdeH is a diguanylate cyclase that is involved in producing the key secondary messenger c-di-GMP. c-di-GMP is proposed to be a "master regulator" of biofilm formation.

Its expression increases surface adhesion and various aspects of the biofilm response.

We use this and others diguanilates (Wspr, YddV) in petSUMO plasmid to run the test. We did experiment with and without agitation, and also testing the efficiency of biofilm in coconut fiber.

In our test YdeH appeared to be very efficient in biofilm production and probably ideal to use in our circuit as YddV.

YddV

Submitted by another iGEM team, iGEM12_HIT-Harbin, (the fragments codes for YddV, a diguanylate cyclase originated from Escherichia coli O7:K1 genome).

We have been running tests of biofilm expression and growth rate of this protein in petSUMO vector intending to add new information about an existing part.

We use this and others diguanilates (Wspr, YdeH) in petSUMO plasmid to run the test. We did experiment with and without agitation, and also testing the efficiency of biofilm in coconut fiber.

In our test YddV appeared to be very efficient in biofilm production and probably ideal to use in our circuit as YdeH.


PA3702 (Wspr)

This part is originated from Pseudomonas aeruginosa. In our project we use this Wspr to test biofilm quantification in order to see what diguanilate is more efficient for our circuit.

We use this and others diguanilates (Yddv and YdeH) in petSUMO plasmid to run the test. We did experiment with and without agitation, and also testing the efficiency of biofilm in coconut fiber.

In our results we conclude that Wspr is the less efficient diguanilate when compared to YdeH and YddV.

MAP

LINEAR


RBS + tDGC

This one is originated from Thermotoga maritima

Cyclic di-GMP (c-di-GMP) is recognized as an intracellular signaling molecule coordinating the “lifestyle transition” from motility to sessility and vice versa (i.e. dispersion).

The correlation between high c-di-GMP concentration in the cell and biofilm formation or between low c-di-GMP levels and motility has been demonstrated in several bacteria species [1].

MAP

LINEAR

MERCURY SYSTEM

MBP (Metal Binding Peptide) + Linker

This part has a segment of BBa_k346004. We added a short linker. MBP is a lead metal-binding peptide engineered from PbrR.

This part is a protein domain and increase levels of c-di-GMP to induce biofilm formation.

MerR + Bidirectional Promoter

Terminator tpr + MerR+ bidirectional Promoter + RBS (Mer operon)

This part is a segment of BBa_K1355001. This biobrick has a bidirectional promoter, having dual functions: A) In reverse: transcription of the MerR regulator protein; and B) In forward: opened for insertion.


LINEAR MAP


GRAPHS

Below we can see some results for biofilm formation, it's possible to see in detail on the RESULTS page.

SECRETION MACHINERY

HlyA (BBa_K554002)

HlyA it’s a signal peptide found at α-hemolysin sequence’s C-terminal, this peptide signals for type I secretion of gram-negative bacteria. The attachment of signal peptide HlyA to the target protein, mediated by HlyB-D complex, must result in the protein transportation to the extracellular medium in a single step process. When the ligation process ends, the HlyD will induce the interaction with secretion machinery protein TolC. For the secretion process to happen, we only need the last 50-60 aa of c-terminal region of HlyA.

HlyB (BBa_K554007)

Responsible for secretion process specificity due to substrate recognition.

HlyD (BBa_K554008)

Adaptation protein, has a cytoplasmic domain followed by a membrane anchor and a large periplasmic domain.

TolC (BBa_K554009)

Channel protein located between the periplasmic region and the extracellular medium.