Team:WashU StLouis/Parts

Washington University - Penn State iGEM

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WashU and Penn State   iGEM 2015
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RBS Characterization

Name Part Number
Artificial RBS for nifZ BBa_K1605002
Artificial RBS for nifE BBa_K1605004
Artificial RBS for nifH BBa_K1605006
Artificial RBS for nifS BBa_K1605008
Artificial RBS for nifK BBa_K1605010
Artificial RBS for nifB BBa_K1605012
Artificial RBS for nifD BBa_K1605014
Artificial RBS for nifN BBa_K1605016
Artificial RBS for hesB BBa_K1605018
Artificial RBS for hesA BBa_K1605020
Artificial RBS for cysE2 BBa_K1605022
Artificial RBS for nifV BBa_K1605024

Composite Part

Name Part Number
Ptet mRFP nifZ RBS BBa_K1605003
Ptet mRFP nifE RBS BBa_K1605005
Ptet mRFP nifH RBS BBa_K1605039
Ptet mRFP nifS RBS BBa_K1605009
Ptet mRFP nifK RBS BBa_K1605040
Ptet mRFP nifB RBS BBa_K1605013
Ptet mRFP nifD RBS BBa_K1605015
Ptet mRFP nifN RBS BBa_K1605017
Ptet mRFP hesB RBS BBa_K1605019
Ptet mRFP hesA RBS BBa_K1605021
Ptet mRFP cysE2 RBS BBa_K1605023
Ptet mRFP nifV RBS BBa_K1605025

Promoter Characterization

Name Part Number
High constitutive expression cassette BBa_K314100

RBS Composite Parts

The BioBricks we submitted to the Registry were composite parts composed of a constitutive pTet promoter part BBa_R0040; individual RBSs designed for use in our minimal nif plasmids; mRFP part BBa_E1010; and two terminators, parts BBa_B0010 and BBa_B0012. They were derived from the BBa_I13521 plasmid. We replaced the RBS BBa_B0034 on that plasmid using PCR primers attached to our desired RBSs.

After constructing our plasmids, we ran PCR of the regions on the plasmids that were to be sequenced and obtained the proper lengths (approximately 1200 base pairs). We then sequenced-verified the regions on our plasmids. Finally, we ran a fluorescence experiment in which we examined the relative amount of mRFP fluorescence with our artificial RBSs as compared to the original RBS present in part BBa_I13521. Our RBSs did not express the mRFP as highly as the original RBS, which was expected. The data we obtained fit our translation initiation rate predictions fairly well. We obtained an R²-value of .9061.

Promoter Characterization

The 2015 WashU iGEM conducted a series of induction experiments to determine the validity of part K314100. The results obtained suggest that the part isn’t subject to inducer concentrations. The part either fluoresced at a constant, high level or didn’t fluoresce at all when tested.

The team transformed the part into DH10B twice. The colonies of the first transformation looked like this:

The cells largely appeared red. The WashU iGEM team then conducted an induction experiment to test how the cells fluoresced at different concentrations of aTc. The team pipetted cell culture into different aTc concentrations serially diluted across 8 wells. Results from this induction experiment are shown below.

While the part had a 1.3x increase between the lowest and highest inducer concentrations, the fluorescence increase was not uniform across all concentrations; fluorescence decreased at certain concentrations when it should’ve been increased.

The team proceeded with a second transformation to test the part even further. Colonies from that transformation are shown below.

Cells were largely colorless, with few red colored colonies to be found. Using the same procedure from the first experiment, the team ran a second induction experiment on these colonies to observe whether red fluorescence increased with aTc concentration. The results from that experiment are shown below.

The colonies exhibited no remarkable fluorescence regardless of the aTc concentration. At this point in time, the team hypothesized that the colonies from the first transformation were simply different than the colonies from the second transformation, perhaps as a result of homologous recombination. The team decided to run a third induction experiment, with the same protocol used in the previous two experiments, to determine the validity behind this assertion. Two colonies were picked from the first transformation; one colony was picked from the second transformation. The results from this experiment are shown below.

Values for each colony are consistent with what was obtained in their respective experiments. While the team didn’t sequence the parts in each colony, it suspects that two separate strains had been produced, in which the part had been mutated in some manner. Regardless, the team holds little confidence in the effectiveness of this part.