Olmec 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

To build an oscillator out of genetic components that exhibits better autocorrelation than the repressilator.

Description of SystemWe designed 3 new repressor-promoter pairs (all from the lamboid cI family - 434 cI, P22 c2, and HK022) in order to make a double oscillator - two linked repressilators, each controlling its own reporter (HK022 was chosen to be our 'spare part'). Theoretically, the two rings will synchronize each other and stabilize the oscillations of the reporters over time.

Theorized Biological Mechanism

System Design

Parts List

BBa_B0002 - Base Plasmid for Structured Assembly with link to Actual Biobricks page

BBa_B0011 - Transcriptional Terminator 2

BBa_B0012 - TE Transcriptional Terminator from Bacteriophage T7

BBa_B0034 - RBS-5 Elowitz RBS

BBa_C0012 - LacI Protein LVA w/o RBS

BBa_C0040 - TetR coding region LVA

BBa_C0050 - cI coding region from HK022cI- LVA

BBa_C0051 - cI coding region from Lambda - LVA

BBa_C0052 - cI coding region from 434

BBa_C0053 - c2 coding region from p22 -LVA

BBa_E0022 - CFP w/o RBS w/ LVA

BBa_E0032 - YFP w/o RBS w/LVA

BBa_R0011 - IAP Inverting regulator driven by LacI (BBa_C0010, 11)

BBa_R0040 - Inverting regulator driven by C0040 (TetR)

BBa_R0050 - cI Regulatory Region

BBa_R0051 - cI regulator from Lambda

BBa_R0052 - Regulator region driven by C0052 (cI)

BBa_R0053 - Regulator region driven by C0053 (c2)

 

Methods

Simulations

We performed several simulations using Matlab (Continuous) and Stochastirator (Discreet) to test the expected performance of our system. The results and setup is discussed below. 

Summary

As discussed above, the system is setup as a a double oscillator, with each system sharing a common protein. Since there were six proteins to choose from, and five positions in which they could be located, more than 7000 permutations are possible. To choose the best performing systems it was necessary to simulate each permutation in a systematic manner and order each system according to some ranking values, such as stability of phase, frequency, and amplitude. 

Graphs

Link to Graphs here. 

Source Codes

The Source Code of the Scripts is above. 

Important Parameters (and Links to Articles from which gathered)

K-Ds, etc

Design Issues

Initially, the Elowitz RBS (BBa_B0034) will be used with all coding sequence components.  This RBS can be adjusted if necessary based on results from debugging experiments (see BBa_B0030 - RBS-1 Strong, BBa_B0031 - RBS-2 Medium, BBa_B0032 - RBS-3 Weak, BBa_B0033
RBS-4 Weaker).

To ensure termination of transcription, a double terminator will be used after every coding sequence (BBa_B0012 - TE Transcriptional Terminator from Bacteriophage T7 followed by BBa_B0011 - Transcriptional Terminator 2).

Since the phage repressor regulatory regions include partial promoter sequences driving transcription on the reverse strand, a bidirectional terminator will be placed upstream to prevent undesirable transcription events.

HK022 was chosen as the spare part for two reasons: one, the least is currently known about its possible interactions with other phage repressor/promoters, and two, it has a significantly higher degree of cooperativity than the other lamboid repressors, so it is most likely to unbalance the rings.

Another design issue is plasmid copy number: we are placing our system on low-copy plasmids, but the reporters on high-copy in order to create stronger YFP and CFP signals. This design may result in over-dilution of repressor proteins (tetR and LacI, specifically), however.

Challenges and Debug Plan

Potential problems:

1.  Cross-talk between different repressor-promoter pairs.  This may lead to either stable or chaotic behavior (i.e. not oscillatory).

2.  The different repressor-promoter pairs are imbalanced with respect to either repressor or promoter strength.  According to simulations, this may lead the two oscillators to have sufficiently different periods such that synchronization is impossible.

Debug Plans:

1.  Cross-talk experiments

Place each repressor under the control of an externally-controllable promoter (such as LacI or tetR) and each regulatory region in control of YFP.  Each repressor and regulatory region can be tested in a combinatorial pair-wise fashion to assess both cross-talk and the tightness of repression (if the repressor is used in conjunction with its corresponding regulatory region).  These experiments have the additional benefit of establishing transfer curves for each repressor-promoter pair.  This leads to a total of 36 parallel experiments.

2.  Balance experiments

Much of the information concerning the relative strengths of the promoter-repressor pairs can be established in the experiment detailed above.  However, an additional debugging strategy may be to take the repressilator and swap each of the three new phage repressor-promoter pairs for the lambda cI repressor-promoter pair to establish whether there is oscillation and if so, how its frequency compares to the repressilator.  The same experiment can be repeated in a phage-derived repressilator to compare lacI and tetR.

3. Real-Time Debug

Since the double oscillator is set up so that tetR and LacI are in separate rings, each ring can be separately disabled at any time during the experiments with either IPTG or ATC, if necessary.

Future Research

An interesting idea to pursue is to make the repressilator with a protease inserted at one step in addition to the repressor.  The corresponding component in the repressilator could be degraded specifically by this protease in order to achieve a smaller period.  The TEV protease is an ideal candidate for such a project.  

References

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

 

Group Members (Include Picture of Group)

Reshma  Shetty

Maia Mahoney

Deva Seetharam

Jose J Pacheco