Part:BBa_K374006:Experience

Characterization of BBa_K374006

Positive feedback antitermination on double reporter plasmid

The iGEM DTU team 2010 have submitted this part, after using it in the development of a genetic bistable switch.

Aim and strategy

The aim of the characterization was to asses if this part together with BBa_K374005 could have an antiterminating function that could be used as a regulatory mechanism. We characterized the functionality of the part in the following construct.

The test plasmid was constructed to biobrick standards, for more information see our wiki, GFP and RFP was used as reporter proteins. The distance from the boxB site in the nut site (marked with a grey triangle) to the terminator was 26bp. The RBS sites was the BB standard RBS BBa_B0034, except for the N protein where the natural RBS site was used. 5 constructs where produced named from A to E, with three different terminator strengths and references with out terminator and N protein.

we tested if:

• If it was possible to trigger a positive feedback mechanism of N protein, by varying the promoter strength with the synthetic promoter library (SPL) and thus antitermination and constant expression of the down stream RFP.
• If it was possible to trigger this positive feed back mechanism by inducing the N protein from a second plasmid insert with the pBAD promoter and the N protein.

The following test strains were constructed, see the table below. All the constructs was transformed with the a synthetic promoter library, to gain a wide arrange of promoter strength to test the effect and functionality of the constructs. (see the Biobrick standard on synthetic promoter libraries SPL - http://bbf.openwetware.org/RFC.html#BBF_RFC_63:_DTU_Synthetic_Promoter_Library_Standard BBF RFC 63) Of the original colonies selected from the transformation of Serie A constructs. 11 were selected for transformation with pAT01, the inducible pBAD+Nprotein plasmid. This series of 11 strains were succesfull transformed with pAT01, serie N.

The table below contains the different strains constructed used in the characterization experiments.

Strain code	Serie A	Explanation	Serie N
A	pAT12	no terminator	+ pAT01
B	pAT13	strong term. B0015	+ pAT01
C	pAT14	medium term. B0011	+ pAT01
D	pAT15	weak term. B1003	+ pAT01
E	pAT16	No N protein, weak term.	+ pAT01

All the strains were tested both with florescence microscope, flourometer and a microfermentor system.

Results

In this section is listed the results from the different verification experiments.

Florescence microscope Flourescence microscope was used to investigate the success rate and verify the preformed transformations with the SPL. We looked at the first transformations done with the test constructs and the SPL, and selected 10 colonies from each construct for further analysis. The colony pictures is presented in the figure below.

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following the first transformation we did a series of restreaks of the successful serie A transformations described above. These were used for Biolector experiments, following we looked at the colonies in the microscope to verify BioLector data and construct stability.

The efficiency rate were drastically decreased, as was the consistency regarding the terminator efficiency. Most of the colonies that had undetectable levels of GFP have gained a mutation that prevents expression of a functional GFP, as they had detectable levels when isolated, expect two colonies in each construct that was selected for low GFP expression. Only one colony (B10) of the selected and restreaked colonies from the B and C constructs expressed trigger mechanism and expressed RFP, see the table below.

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Micro fermentor Overnigth cultures of both series A and N were run in the Micro fermentor and serie N were induced with arabinose after approx. 4 hours to induce N protein expression, to verify if the trigger mechanism seen from the florescence microscope (see below) could be induced. Of the 9 cultures only one of the E-constructs gave a GFP signal (strong promoter – weak Terminator – no N protein), but non of the constructs expressed RFP. Data not shown.

Fluorometer At the same time the overnight cultures were diluted and new overnight cultures were made with a reference and also induced with arabinose to test if a difference in the RFP expression could be seen. Non of the cultures expressed RFP. Data not shown. But it could clearly be seen on the cultures, by visually inspection, that the strains that had N induced had growth drastically decreased, and seemed to be lysing, as reported in the litterature.[1]

Conclusionc and Recommendations

From the initial verification by florescence microscopy, our constructs seems to work and have the expected functionality. It was though not possible to verify the function and mechanism in the subsequent micro fermentor experiments.

We expect that this biobrick works, and that it together with BBa_K374005 can induce anti-termination. We recommend that further verification should be done with different reporters as the florescence protein clearly stresses the cell and show a high degree of mutation. Constitutive expression of the N protein should be avoid as it have severe effect on the cells when expressed in high levels.

For the mechanical engineering of an anti-terminator system, the distance between the nut site and the terminator should be investigated to find the minimum distance that still allow antitermination. We used a distance of 26bp in our constructs, due to the reporter system, we cannot conclude that this does not work, but it is a possible area for optimization of the antiterminator mechanism.

references

• [1] Naomi. C. Franklin., Jed. H. Doelling., (1988) "Overexpression of N Antitermination Proteins of Bacteriophages A, 21, and P22: Loss of N Protein Specificity" Journal of Bacteriology.

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BBa_K374006 1 Not understood iGEM DTU 2010

We have characterized this part.

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