Difference between revisions of "Part:BBa B0015:Experience"

iGEM Athens 2019

We attempted to measure the efficiency of the terminator by cloning it into the pSB1A10 plasmid and culturing the bacteria for 24 hours (37 degrees Celsius, 200RPM, 0.1% arabinose, 100 µg/mL ampicillin). The culture was pelleted and the pellet was resuspended in 1mL of 1X PBS. From that, 10 μL were diluted in 990 mL of 1X PBS to yield approximately 5 million cells. For FACS, we used BD FACSCanto™ II. During FACS, a low flow rate was used. We used the following excitation and emission wavelengths: GFP: excitation = 488 nm, emission = 510 nm ; RFP: excitation = 488 nm, emission = 584 nm.

FACS measurement. The terminator was named EKATI for our project.

The efficiency is measured through the following formula: Termination efficiency = 1 -{[RFPterm/GFPterm]/[(RFPcontrol/GFPcontrol)mean]}. This measurement showed that the efficiency of the terminator is 77.4%. We also measured the terminator efficiency using the GFP+RFP+ population and the formula: Terminator efficiency = 1 - (Intensity of GFP+RFP+term/Intensity of GFP+RFP+control). This measurement showed that the terminator efficiency of the terminator is 82%.

iGEM Team Göttingen 2013

We used the part for our reporter system and it worked very good! We also improved it by switching the pre- and suffix, basically inverting it. This way, we were able to use it in an "inverted" expression unit on the same vector as our reporter system. For further information see: https://parts.igem.org/wiki/index.php?title=Part:BBa_K1045009

iGEM13_Fudan

Background

Csy4 is a member of CRISPR pathway discovered in Pseudomonas aeruginosa. It is a single endoRNase that recognizes and cleaves a 28-nucleotide repetitive sequence and produces stable transcripts with a 5’hydroxylgroup, which can eliminate unwanted interactions between 5’UTRs and translational elements such as RBSs units to standardize the expression of the elements. ( Lei Qi et al, 2012).The cleavage site can be curtailed to 16nt proved by in vitro data (Rachel E. Huarwitz et al, 2010)

The CRISPR RNA-processing system facilitate engineering of standard genetic elements in various contexts( Lei Qi et al, 2012).

Figure. 1. The CRISPR RNA-processing system facilitate engineering of standard genetic elements in various contexts( Lei Qi et al, 2012).

Design

Csy4 system is introduced into the terminator efficiency test assay and used optimized sfGFP to test the PoPs of Ptet (BBa_R0040), to approximately estimate the input of BBa_B0015. Then we tested the output of BBa_B0015, with Csy4 insulator to avoid the RNA interference.

Construction for termination efficiency estimation

Figure A. Construction for termination efficiency estimation. We used optimized sfGFP to test the PoPs of Ptet (BBa_R0040), to approximately estimate the input of BBa_B0015. Then we test the output of BBa_B0015, with Csy4 insulator to avoid the RNA interference to sfGFP translation.

Experimental Data (Quantitative Measurement)

We get the device measured by flow cytometer by testing the expression of sfGFP.

Histogram of the Flow cytometer measurement

Figure B, Histogram of the expression of output of terminator, measured by sfGFP fluorescence by flow cytometer. We assembled the promoter BBa_R0040 and the Csy4 protein with the terminator BBa_B0015. And we used the sfGFP protected by a insulator to measure the leakage rate of the terminator.

Then, we found a decrease of terminator efficiency to only 0.657 (Figure C), due to the downstream interference, largely inconsistent with the previous study that showed that BBa_B0015 is a strong terminator, with 0.984[CC], 0.97[JK]，forward efficiency, of which the rrnB_T1 (B0010) part also has 97.27% forward efficiency (G. Cambray, et al., 2013).

Measurement of BBa_B0015 efficiency

Figure C, Measurement of BBa_B0015 efficiency: column of the median values of fluorescent expressions with/without the terminator and the interaction between the csy4 and the 16-nt cleavage site. (Left: Input/Right: Output) The error bar represents two quartiles.(Up:75%, Down:25%) The terminator efficiency is (1-output value/input value).

Reference:

Haurwitz R E, Jinek M, Wiedenheft B, et al. Sequence-and structure-specific RNA processing by a CRISPR endonuclease[J]. Science, 2010, 329(5997): 1355-1358. Qi L, Haurwitz R E, Shao W, et al. RNA processing enables predictable programming of gene expression[J]. Nature biotechnology, 2012, 30(10): 1002-1006. Cambray G, Guimaraes J C, Mutalik V K, et al. Measurement and modeling of intrinsic transcription terminators[J]. Nucleic acids research, 2013, 41(9): 5139-5148.

Aberdeen_Scotland 2009

The transformation, Plasmid miniprep and gel (undigested and digested with Xbal and PstI) worked as expected.

Michigan iGEM 2012

As previously observed by other teams, when BBa_B0015 is PCR amplified using VF2 and VR unexpected bands of varying lengths (a bright band at 500bp and several dim ones below 500bp) appear on the electrophoresis gel.

Aberdeen_Scotland 2009

The transformation, Plasmid miniprep and gel (undigested and digested with Xbal and PstI) worked as expected.

Michigan iGEM 2012

As previously observed by other teams, when BBa_B0015 is PCR amplified using VF2 and VR unexpected bands of varying lengths (a bright band at 500bp and several dim ones below 500bp) appear on the electrophoresis gel.