iGEM 2018 — Team Michigan

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# Team:Michigan/Notebook

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2014 ATOMS-Turkiye](https://2014.igem.org/Team%3AATOMS-Turkiye/Notebook)
* [2014 Tec Monterrey](https://2014.igem.org/Team%3ATec-Monterrey/ITESM14_project.html#tab_notebook)
* [2014 Kyoto](https://2014.igem.org/Team%3AKyoto/Notebook/Magnetosome_Formation#title)
* [2014 Cornell](https://2014.igem.org/Team%3ACornell/notebook)


[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Michigan/Experiments

Michigan:Attributions

## Experiments

### Building the Attack/Guard Assay:

SaCas9, gRNA for SaCas9, and gRNA for SpCas9 were synthesized by Integrated DNA Technologies. SaCas9 was synthesized from two linearized fragments, with a Ava restriction site near the 5’ end of one and 3’ end of the other to join them into one fragment. This complete fragment was flanked by EcoRI and PstI sites in order to be compatible for insertion into the submission vector pSB1C3. Once the SaCas9\_C plasmid was confirmed, we double digested the SaCas9\_C and the gRNA for SaCas9 with SacI and SgrAI and then ligated the fragments, leaving us with one of our final plasmids needed for experiments, SaCas9\_C\_gRNA, a plasmid containing a LacI coding region for inducible control, the gRNA able to transcribe and bind with SaCas9 in order to target the complementary sequence on the reporter plasmid, and the SaCas9 coding region.
![](https://static.igem.org/mediawiki/2018/9/98/T--Michigan--SVG1.svg)

We obtained the SpCas9 DNA from the Zhang lab at the University of Michigan in a pET083 backbone. In order to move the fragment we needed (containing LacI repressor and SpCas9) into pSB1K3, we used PCR mutagenesis to flank our region of interest with EcoRI and PstI cut sites. Once SpCas9\_K was confirmed, we double digested with the gRNA for SpCas9 using sites EcoNI and SgrAI. Then we ligated the resulting fragments, leaving us with our final plasmid needed for experiments, SpCas9\_K\_gRNA, a plasmid containing a LacI Repressor coding region for inducible control, the gRNA able to transcribe and bind with SpCas9 in order to target the complementary sequence on the reporter plasmid, and the SpCas9 coding region.

![](https://static.igem.org/mediawiki/2018/a/a7/T--Michigan--SVG2.svg)

Given more time to conduct experiments, we would induce the necessary point mutations in each Cas9 through the use of One-Pot Mutagenesis (see protocol section). This requires primers be designed to anneal to the specific portion of the the sequence where we want to induce our mutations. These mutations will be specific to each Cas9 used since the nuclease domain will be in a different location within the sequence in each case.

The reporter plasmid (with CFP) was synthesized by Integrated DNA Technologies to contain the necessary target sequence and PAM sequence needed for recognition of SpCas9 and SaCas9 with their respective gRNAs.

All restriction digests were performed using New England Biolabs (NEB) restriction enzymes. Qiagen PCR cleanup kits were then used on each restriction digest product. All ligations were performed with NEB T4 ligase at room temperature overnight, then the ligation products were first transformed into homemade DH5a chemically competent cells (see protocol section), but after a couple failed transformations, we used NEB DH5a chemically competent E. coli cells. These transformations were plated and grown overnight. 5mL liquid cultures were prepared from colonies picked from the plates the next day and themselves grown overnight. All DNA extraction was performed using Qiagen miniprep kits. CFP was measured at 398 nm excitation and 498 nm emission via a plate reader (Infinite 200, Techan Life Sciences). Cell density was measured at A600 via the same plate reader.

* Project
* Experiments
* Results
* InterLab Study

* GACCA
* Project
* The Team
* Human Practices
* Parts
* Modelling
* Safety
* Collaborations
* Attributions

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Michigan/Results

Michigan:Results

## Results

Control: Reporter plasmid in E.Coli - Will we be able to see fluorescence in cells with the reporter plasmid CFP.

### Figure 1: Fluorescent images

![](https://static.igem.org/mediawiki/2018/4/47/T--Michigan--CFPC1.jpg)
![](https://static.igem.org/mediawiki/2018/6/6a/T--Michigan--CFPC1_3.jpg)

We were able to show that E.coli transformed with the reporter plasmid expressed for the CFP gene as expected, although the level of fluorescence/expression was relatively low.

Experiment #1: Will SpCas9 + Reporter and SaCas9 + Reporter effectively degrade the Reporter plasmid, reducing CFP over time?

Unfortunately, due to our struggles with cloning and assembling our constructs, we were unable to perform any of the experiments we had planned, other than confirming expression of our reporter. Although we were able to see fluorescence in our reporter control through imaging a previous time, the control reporter during the experiments did not express enough CFP for comparison of time points and see further reduction.

We were unable to move forward with the rest of the experiments before the wiki freeze due to time constraints, issues with the double transformation required by the first experiment, and problems with SaCas9 and SpCas9 point mutagenesis to form respective dead Cas9s. We are continuing to work to complete this project and hope that we are able to present results from our planned experiments at the 2018 iGEM Jamboree.

#### Conclusion:

We were able to show fluorescence with the reporter plasmid; however, we did not have enough time to get any results from experiments. It is difficult to extrapolate meaningful results especially given the low level of CFP expression in the control, beyond basic observations that the reporter plasmid was successfully transformed and its reporter gene expressed to some degree. In the future, establishing a viable control may be facilitated through the use of competent cells which are optimized for protein expression (BL21), or by creating recombinant plasmids based on high-copy number vectors (although this would still have to be compatible multi-plasmid transformation). This would hopefully allow for more demonstrable and observable changes in CFP expression when cleaved by the “assassin” Cas9 protein, and serve as a baseline for fluorescence when blocked by the “guardian” Cas9.

#### Future Directions:

Given more time, we would establish controls that demonstrate baseline fluorescence, the ability of nuclease active Cas9 to cleave the target plasmid, and the ability of deactivated Cas9 (dCas9) to bind but not cut the target sequence. Once these controls are established and standardized, we would then move into the competition experiments. Working with the SaCas9 and SpCas9 systems we expect to see a considerable difference in the relative binding kinetics and affinity, giving us a clear winner in the competition. This data will then be incorporated into our mathematical modeling to show whether or not our predictions were correct and calculate a quantitative measure of relative binding affinity and kinetics that will be able to be compared between experiments. Following this process of validating the basic protocol, we will attempt to engineer a Cas9 enzyme to have some desired property and set up this experiment to compare the new design to the WT Cas9.

* Experiments
* Results

* GACCA
* Project
* The Team
* Human Practices
* Parts
* Modelling
* Safety
* Collaborations
* Attributions