N*SYNCH Group Page

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2003 Groups

N*Synch

Olmec

Young Power

Prakash

Biobricks

 

Design Goal Methods References
               

Applications Link; This link will go to a  common page for all groups describing why we did these projects. (Do not work on this for each group, we'll take care of it.

Background
For more information about Synthetic Biology visit the Synthetic Biology web pages at MIT.

For more information about Biobricks visit the Biobricks page.

For more information about the repressilator system see this article.  

Design Goal

Our goal was to build an XOR gate using antisense inhibition strategies. The output, and one of the inputs of the XOR can be interfaced with the repressilator to yield two possible types of behavior. If the external input of the XOR gate is set to a low state, the system will function as a repressilator. If the input is set to a high state, the system will hold its current signal values and therefore function as a memory latch.

Description of System

Theorized Logic Mechanism

Below is a simple schematic of an XOR gate. A "0" refers to the absence of a signal (e.g. a protein or ligand), while "1" refers to the presence of some signal above a certain threshold (to be determined later).

The XOR can be interfaced into the repressilator, as shown below.

 

The logic table of this system is given below for three time steps (a time step is the amount of time necessary for a signal to propagate through one cycle of the repressilator). It is assumed that at t= 0, the levels of A, B, C are already determined. Input D is added at t= 0, and each subsequent time step reveals the states of A, B, C, and D.

Table 1: For D = 0, the system behaves as a repressilator

Signal (at output terminal)
t= 0 time steps
t = 1 time steps
t = 2 time steps
A
1
0
1
B
0
1
0
C
1
0
1
D
0
0
0

Table 2: For D = 1, the system behaves as a latch that stores initial values

Signal
t= 0 time steps
t = 1 time steps
t = 2 time steps
A
1
1
1
B
0
0
0
C
1
1
1
D
1
1
1

 

Biological Implementation

To build this XOR using biology, we used the following components:

BB ID#
Usage (gates)
Description
BBa_B0011
A, B, C
Bidirectional transcription terminator
 
BBa_B0012
A, B, C
TE transcriptional terminator
 
BBa_B0034
A, B
RBS-repressilator
 
BBa_C0012
input
LacI Protein LVA w/o RBS
 
BBa_C0040
input
TetR coding region LVA
 
BBa_E0022
A, B, C
CFP w/o RBS w/ LVA
 
BBa_E0032
A, B, C
YFPw/o RBS w/LVA
 
BBa_R0040
input
Inverting regulator driven by C0040 (TetR)
 
BBa_R0051
A, test module
cI regulator from Lambda (repressillator)
 
BBa_R0053
input
p22 Inverting regulatory driven by c2 (BBa_C0053)
 
BBa_I1010
C
RNA regulation target
( Promoter driven by C0040 and cI mod #1 CDS)
 
BBa_I1033
C
I1010 interferer (IS10)
 
BBa_I1020
C
RNA regulation target
(Promoter driven by C0062 and cI mod #2 CDS)
 
BBa_I1023
C
I1020 interferer (IS10)
 
BBa_I1030
C
I1010 with Junk instead of Loop
 
BBa_I1031
C
I1030 interferer (KISS)
 
BBa_I1032
C
I1030 interferer (micRNA)
 
BBa_I1034
C
1031, no promoter
 
BBa_I1040
C
I1020 with Junk instead of Loop
 
BBa_I1041
C
I1040 interferer (KISS)
 
BBa_I1042
C
I1040 interferer (micRNA)
 
BBa_I1044
C
I1041, no promoter
 
BBa_I1060
C
I1020 with 4 stem swaps
 
BBa_I1063
C
I1060 interferer (IS10)
 

Note that when the XOR is interfaced with the repressilator, we will need to use additional respressilator parts (hopefully to be characterized by OLMEC group).

The key biological constructs in our system are shown in the diagram below:

 

Below are the pieces we'd like preassembled (if Blue Heron will do this for us)

 

The XOR gate, with its key elements, are organized into a represillator circuit shown below:

 

Description of system with XOR gate inserted into a represillator. This illustration shows the XOR genetic elements integrated into a repressilator network. The function of the XOR gate depends on 2 inputs. IPTG upregulates the expression of genes controlled by the PLlacO-1 promoter, while aTc upregulates the expression of genes controlled by the PLtetO-1 promoter. The promoters are repressible by lacI and tetR, respectively, and the input chemicals IPTG and aTc function by binding the repressor proteins and therefore preventing their interaction with their promoters.

Antisense RNA is a mechanism for preventing the expression of protein at the level of mRNA. In general, antisense strands function by specifically targeting mRNA that is complementary in its base pair sequence. Details of the antisense mechanisms, and a description for the mechanisms we employ, are provided below.

The XOR consists of 2 ‘branches.’ Within each branch a coding region for the cI protein is flanked by an antisense element. The antisense element is designed to inhibit its target on the other branch, such that if both inputs to the XOR gate are active (promoters are in the ‘on’ state, the antisense elements will block the expression of cI protein. Having two distinct antisense-target combinations requires that we make one coding sequence for cI different than the other, such that the antisense element does not target non-specifically. This accounts for the two versions of cI, cI(1) and cI(2).

If the XOR gate functions as expected, it can be inserted into a repressilator system, where it will function as an oscillating device or a memory storage latch (see above). However, care must be taken in choosing the elements of the repressilator system. Any element containing a lacI or tetR component is not compatible with the XOR.

Antisense Biological Mechanisms

The success of this system clearly rests on the ability to effectively and specifically target mRNA transcripts for degradation using anti-sense RNA. While many papers, articles, and books have been written on the subject, there are no consensus anti-sense building strategies presented. We thus chose to implement three different types of antisense inhibition: KISS, micRNA, and IS10. In the description that follows, the following nomenclature will be used:

target- the mRNA transcript that we wish to inhibit.

antisense- the anti-sense molecule which will bind and inhibit target.

1. KISS (Keep it SImple, Silly)

The simplest of the three methods, this type relies on a single-stranded linear 108 bp antisense that is specific to the target of interest. Transcripts resulting from BBa_I1030 and BBa_I1040 each contain a different "junk" region (34 bp) that is upstream of the RBS. In addition, the first 72 base pairs of the cI region of BBa_I1030 and BBa_I1040 have been codon-modified to give different sequences that code for the same cI protein. These version of cI are called cI(1) and cI(2), respectively. The RBS and start codon regions are the same for both BBa_I1030 and BBa_I1040.

BBa_I1031 and BBa_I1041 contain the reverse complement of the junk regions, RBS, start codon, and initial cI region for BBa_I1030 and BBa_I1040, respectively. Thus, if both BBa_I1030 and BBa_I1031 are transcribed, the transcripts will bind to each other and BBa_I1030 will not be translated. The same is true BBa_I1040 and BBa_I1041.

Note that BBa_I1030 and BBa_I1040 each already contain a regulatory region, RBS, and coding region (a terminator must be added), while BBa_I1031 and BBa_I1041 do not - thus, when using these components, the appropriate regulatory region, RBS, and terminator must be added to these parts.

2. micRNA

The method of middling difficulty, this type of anti-sense relies on two stem loops flanking an anti-sense sequence that is specific for the target. The function of the stem loops is to maintain the antisense region in a quasi-linear state. BBa_I1032 and BBa_I1042 are built in this manner, with a linear region that will bind to the junk, RBS, start codon, and initial cI regions of BBa_I1030 and BBa_I1040.

 

 

3. IS10

 

This method is modeled after the mechanism by which IS10 inhibits production of IS10 transposase. The anti-sense strand is transcribed from the complementary strand of the target (figure above), resulting in an anti-sense strand that is 115 bp long, of which 35 bp is complementary to the target. In the absense of target, these 35 bp form a weak stem loop with the rest of the anti-sense molecule (figure above). The key element of the system is the loop at the tip of this stem loop (C-G-G-C-U-U...), which is held in a linear state by the rest of the loop. Upon exposure to the target, the linear loop is able to bind to the 5' end of the target (G-C-C-G-T-T...), and initiate an energetically-favorable zipping/twisting-together of the target and the 5' end of the stem loop (figure below). In other words, one side of the weakly stable anti-sense stem loop binds 35 bp of the target, to form a more stable duplex.

Here are the different permutations of IS10 designs used for this project:

I1010 and I1013

Bio bricks part BBa_I1013 codes for the exact antisense stem loop used in IS10, with two base changes. The 5'-most residues from IS10 anti-sense transcript ( U-C), which do not form part of the stem loop, were changed to G-A. These two bases are reverse-complementary to the first two base pairs of the wildtype cI(1) coding region of BBa_I1010, and thus can bind this region. The rest of the stem loop is wild-type.

The BBa_1010 transcript is targeted by BBa_I1013. The first 35 bases at the 5' end of BBa_I1010 are identical to the first 35 bases at the 5' end of the wild type target, with two differences. The two residues at 114 and 115 on the target in figure above are changed from G-A to T-C, which are the first two bases in the wild-type coding region for cI(1). Note that three bases (T-G-C), which code for cysteine, have been inserted at the 5' end of the cI(1) coding region, before this T-C. This allows us to use a wild-type binding pattern at the base of the stem.

I1020 and I1023

To create a second target/anti-sense system using the IS10 method that would not interact with the BBa_I1010/BBa_I1013 system, we created parts I1020 and I1023. These parts are identical to BBa_I1010 and BBa_I1013, respectively, except for a swap at position 83 in the stem loop in the figure above. A "swap" refers to switching a set of paired bases-- for example, a G-C could be swapped to become C-G. In this case, the base at position 83 of the antisense (BBa_I1023) was changed from a C to a G, while the corresponding base at position 83 of the target (BBa_I1020) was changed from a G to a C. According to Kittle et al (1989), this swap should cause an 2x increase in association rates between target (BBa_I1020) and anti-sense (BBa_I1023), while at the same time having a low rate of BBa_I1023/BBa_I1010 and BBa_I1013/BBa_I1020 nonspecific binding. Indeed, this "low rate" is 20x below that of specific binding.

I1060 and I1063

To create a third target/anti-sense system using the IS10 method, we created parts I1060 and I1063. These parts are identical to BBa_I1020 and BBa_I1023, respectively, except for the presence of four new swaps in addition to the position-83-swap. These new swaps occur within the stem loop region, to yield the following changes:

Position (figure above)
Left half of stem
Right half of stem
Target
97
G -> C
C -> G
C -> G
104
G -> C
C -> G
C -> G
106
U -> A
A -> U
A -> U
107
C - > G
G -> C
G -> C

It is unknown how these swaps will affect the stability of the stem loop. For this region, BBa_I1060 and BBa_I1063 are the most experiemental of al the IS10 parts. The specificity of such changes if better known. According to Jain (1995), the changes should cause a decrease in non-specific binding of 4x, 8x, 4x, and 4x, respectively, as measured by the MCI factor. Thus, this should yield a net decrease of 512x.

Methods

Simulations

We created a simulator of our XOR gate interfaced with the repressilator: antisense.m and xorillator.m. When run withexternal ligand concentrations of 0 and 1, the following outputs were produced:

 

Important Parameters (and Links to Articles from which gathered)

Important parameters are outlined below:

The rate of transcription (in transcripts per second)from various promoters was taken fron Elowitz et al.:

k_transcription_placI = 0.5;
k_transcription_ptetR = 0.5;
k_transcription_input = 0.5;
k_transcription_pcI= 0.5;

 

The rate of transcription leakage from various promoters (in transcripts per second) was taken from Elowitz et al. As a tolerance analysis, we found that the system will still function if the leakage rate from the input promoter is equal or less than 18x that of the other promoters.

k_transcription_leakage_placI = 5e-4;
k_transcription_leakage_ptetR = 5e-4;
k_transcription_leakage_pcI = 5e-4;
k_transcription_leakage_input = 5e-4;

 

The following parameters were taken from Elowitz et al:

mRNA_half_life = 120; (sec)
k_mRNA_degradation = log(2)/mRNA_half_life;protein_half_life = 600; (sec)
k_protein_degradation = log(2)/protein_half_life;translation_efficiency = 20; (proteins per transcript lifespan)
average_mRNA_lifespan = 1/k_mRNA_degradation; (sec)
k_translation = translation_efficiency/average_mRNA_lifespan; (proteins per transcript per sec)

 

These parameters, which pertain to mRNA-mRNA behavior, were taken from Jain, C. (1997). Models for Pairing of IS10 Encoded Antisense RNAs in vivo. J. theor. Biol. 186: 431-439.

k_complex = 0.00041; % 1000 * k_translation; (1/(transcript * sec))
k_uncomplex = 0; % 1/sec
k_degradation_complex = 1000 * k_mRNA_degradation; (1/sec)

The following parameters were taken from Elowitz et al:

n_lacI = 2;
n_tetR = 2;
n_cI = 2;
n_input = 2;

KM_lacI = 40;
KM_tetR = 40;
KM_cI = 40;
KM_input = 40;

Challenges and Debug Plan

Testing and debugging the system in 3 not-so-easy steps.

We propose to test the system in a number of steps. Since the success of the project depends upon a functioning XOR gate, we will need to test the function of the XOR gate independently of the repressilator. Efforts will be made to determine the most effective antisense mechanism that 1) inhibits the expression of its target efficiently while 2) minimizing non-specific binding to non-targeted mRNAs.

 

 

 


Our final XOR genetic gate will be constructed using the antisense mechanism that most efficiently and specifically inhibits the expression of its target.

 

Future Research

The nature of future research will depend on the outcome of the present experiments. The antisense mechanism may need modification or even a complete redesign. If the XOR gate works as expected, another area for future research is to use alternate promoters in the XOR gate that would allow its insertion into the original repressilator.

 

References

Usual reference format. If possible include description of each article and link to PDF file in your group folder.

 

Group Members

Grace Kenney

 

Daniel Shen

 

Samantha Sutton

 

Neelaksh Varshney

With special thanks to Austin Che