Coding

Part:BBa_K3136004

Designed by: Liang ZHANG   Group: iGEM19_Shanghai_HS_United   (2019-09-18)
Revision as of 20:53, 27 October 2020 by Lx0402 (Talk | contribs) (Improvement)


FnCas12a-typeIIS

It is a conding sequence of CRISPR-Cas12a from Francisella novicida U112. The sequence is coden optimized.

Improvement by SHSID 2020

Sequence Analysis We blast the sequences of our new part BBa_K3521001 and the existing part BBa_K3136004. As shown in the figure, there is 2852bp inconsistent good, 1058bp inconsistent bad from the old one part.

Figure 7 The Blast Results of BBa_K3521001 and BBa_K3136004

Different Function First of all, we aim to detect different organisms for different illnesses. The old part is used to detect the African Swine fever virus (ASFV). Our new part is used to detect the presence of Listeria monocytogenes, which is easily survival in low temperature like refrigerator environment, and then cause Listeriosis illness, like fever, muscle ache, and so on.

Furthermore, the old part only designed a crRNA that just targeting a site of ASFV, which may have less wide application. In contrast, we design six different crRNAs that can guide our improved Cas12a (FnCpf1) protein targeting six different genomic sites of Listeria monocytogenes, so that our new part can detect more extensive Listeria monocytogenes.



Usage and Biology

BBa_K3136004 FnCas12a-typeIIS

Methods:

1. Put the EP tube containing purified FnCas12a protein at 99℃ water for 15 minutes.

2. After heating, centrifuge the tubes for 5min.

3. Take and install the gel, add the electrophoresis buffer to the sample hole, and check if there is any leakage. Add marker, to the first and last sample holes, add FnCas12a to the other sample holes, respectively.

7. Connect the electrophoresis device to the power supply. Connect the positive electrode to the tank and the negative electrode to the slot for electrophoresis. The voltage is adjusted to 160V.

8. Turn off the power supply and disconnect the electrode until the bromophenol blue reaches the bottom of the release adhesive. Remove the glass sheet from the electrophoresis device and then remove the gel.

9. Soak the gel in Coomassie brilliant blue dye and dye it in a horizontal shaking bed for 15 min.

Observe the protein bands

Characterization of popular BioBrick RBSs

Cas12a detection method:

Materials:

Water. 14.5 μL

Buffer. 2 μL

Enzyme (Cas12a). 0.5 μL

Template. 0.5 μL

(From solution we made after LAMP)

Detector. 2 μL

crRNA. 0.5 μL

(Note: the Deteror is sequence of ssDNA HEX-N12-BHQ1 or FAM-N12-BHQ1 or FITC-T14-Biotin)

Procedure:

1. Add all the materials together:

• Firstly, use pipette to straw 14.5 μL pure water from the pure water bottle to one tube.

• Secondly, use pipette to straw 2 μL buffer to the previous tube.

• Thirdly, use pipette to straw 0.5 μL enzyme to the previous tube.

• Next, use pipette to straw 0.5 μL template to the previous tube.

• Then, add 0.5 μL crRNA to the previous tube.

• Finally, use pipette to straw 2 μL detector to the previous tube.

2. Straw 1 μL of the mixed solution in the tubes to the 96-well Plate.

3. Detect the solution using fluorescent detector machine to check the fluorescence when they react 10min.

Results:

We used two types of fluorescent probe (FAM and HEX) to check the fluorescence of FnCas12a reaction system at 10min as shown in Fig1. Linked to the control and negative samples, the positive sample has a high fluorescence. It means that FnCas12a slices the fluorescent probe in the reaction system and the cracked fluorescent probe produce fluorescence. we also checked the fluorescence of FnCas12a reaction system at 10min by lateral flow using briDetect (Milenia Biotec GmbH) (Fig2).

Characterization of popular BioBrick RBSs
Fig1. The fluorescence of FnCas12a reaction system at 10min
Characterization of popular BioBrick RBSs
Fig2. The fluorescence of FnCas12a reaction system by briDetect

Using fluorescent detector machine, we can detect the fluorescence of FnCas12a reaction system at different time by setting up program properly in the machine. We detect the fluorescence under different concentration FnCas12a and different time(Fig3). With the time growth, the fluorescence gradually raised until a certain time. Higher concentration FnCas12a had higher fluorescence and the fluorescence increased quickly.

Characterization of popular BioBrick RBSs
Fig3. The fluorescence of FnCas12a reaction system in different time.

Reference

Li S Y, Cheng Q X, Liu J K, et al. CRISPR-Cas12a has both cis-and trans-cleavage activities on single-stranded DNA[J]. Cell research, 2018, 28(4): 491.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 310
    Illegal PstI site found at 364
    Illegal PstI site found at 1627
    Illegal PstI site found at 2533
    Illegal PstI site found at 3727
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 310
    Illegal PstI site found at 364
    Illegal PstI site found at 1627
    Illegal PstI site found at 2533
    Illegal PstI site found at 3727
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1253
    Illegal BglII site found at 1586
    Illegal BglII site found at 1706
    Illegal BglII site found at 2000
    Illegal BglII site found at 2444
    Illegal BglII site found at 3335
    Illegal BglII site found at 3425
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 310
    Illegal PstI site found at 364
    Illegal PstI site found at 1627
    Illegal PstI site found at 2533
    Illegal PstI site found at 3727
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 310
    Illegal PstI site found at 364
    Illegal PstI site found at 1627
    Illegal PstI site found at 2533
    Illegal PstI site found at 3727
    Illegal NgoMIV site found at 2480
    Illegal NgoMIV site found at 3319
    Illegal NgoMIV site found at 3530
  • 1000
    COMPATIBLE WITH RFC[1000]


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