Editing the wiki
About the lab
Editing the wiki
About the lab
Purpose of experiment: to continue the backcrossing of these two mutants.
N2 males were crossed to hermaphrodites. 12 F2s were picked for each. 1 F2 were sterile/dead from each mutant.
oLJ49/50 will be used together to genotype unc-7(e5), have to be sent for sequencing. amplicon is 290bp.
oLJ73/74 will be used together to genotype unc-9(fc16). 270bp, digest with HpyCH4V. Only WT will digest to 200+60.
PCR setup and conditions were the same as previous rounds.
#1~#8 of the PCR products for unc-7(e5) candidates were sent for sequencing after exo-sap treatment, oLJ49 being the sequencing primer.
for unc-9(fc16), the digested PCR products were run on a 2% DNA gel. In the right-most lane, PCR product for unc-7(e5) was loaded, just to make sure the PCR was successful before they will be sent for sequencing.
Result for fc16: No6 and 8 were correct. #8 was used for next round of BC.
Note: only worms on #6 and #8 show completely unc. Perhaps in the future rounds of BC, pick by phenotype.
Purpose of experiment: Based on the homeobox paper, the following promoter combinations might achieve RIP specific expression.
ttx-1p: AFD, M2, RIB, RIP; to be cloned in pJL081 (C-term)
ceh-27p: I6, RIM, RIP, RME, RMF, to be cloned in pJL080 (N-term)
However, the bottle neck is that the homeobox paper used translational fusion (fosmid or crispr knock in) and therefore no promoter information available. The problem is that when I checked ttx-1p, I found it contains super long intron immediately downstream of the 1st short exon. In addition, from a paper by Piali Sengupta, https://www.sciencedirect.com/science/article/pii/S0896627301004317 , they said that when they used a translational reporter containing 3kb promoter + all exon and introns, GFP is expressed in AFD neurons and 8 other pharyngeal cells. However, when they use the promoter + 1st exon, it is only expressed in AFD. When they used a fusion that contains the promoter + regions up until exon 4 (including introns), it shows the same expression pattern. This suggest the first three introns also contribute to the correct expression pattern. If we want to recapitulate the expression of ttx-1, we probably have to include this long intron.
Note that I have emailed Sengupta lab, and they think they probably used 2.6kb upstream of ATG. Nonetheless, I used 3kb region.
The following strategy will be used to clone ttx-1p into pJL081.
Step I: oLJ97/98 will first be cloned into GAL4-C. 3kb insert; FseI+AscI.
Step II: Digest the construct with NheI to drop out ~350bp region. This will be used as a backbone for cloning ttx-1p introns+exons.
Step III: oLJ99/100 will be used together to amplify the ttx-1 gDNA (from ATG to part of exon4). 5.6kb. This will be used using gibson assemly with the next fragment (101/102, 0.4kb) as well as the construct of 97/98 in pJL081/NheI). GGSG linker is placed between the exon and GP41-1-C.
Note: I have emailed Dr. Henning Mootz to inquire him about whether it is ok to have some amino acids that are N-term to the intein.
He replied as follows: in general this will work, we do this all the time. It could only go wrong if for some (usually unpredictable) reason the fused sequence interferes negatively with the expression or folding of the intein. If you plan to fuse a folded domain there, I would use an unfolded spacer sequence of maybe 10-20 aa to be on the safe side. We once fused maltose-binding-protein closely to that side of the Gp41-C intein and it didn’t work. With a longer linker it worked again. Although we also have many examples for other inteins in which we fused with no or only a very short linker.
To be on the safe side, I added GGSG linker N-term to the intein.
Note: the final product can be used in combination with ceh-34p in GAL4-N to achieve M2 expression. Alternatively, use this with rab-3p::GAL4-N to see whether the strategy worked.
The above are overall strategies, experiments are written below.
2x Q5 MM 12.5
66 20sec 72 1’30’’ for 97/98, 3min for 99/100, 10sec for 101/102
Use pJL081 as template for 101/102
On 28-10-20, I first used 97/98 to amplify from worm lysis. But instead of 1'30'', I put as 3min in PCR, and I got no band.
Will re-do later.
Purpose of experiment: to amplify sams-5p from genomic DNA and clone into GAL4 vector. Supposed to be MI specific. The genetic facility helped me on many clonings. However, only this promoter cannot be amplified. So I tested using my Q5 polymerase.
2x MM 12.5
A gradient PCR is used.
Result: All correct. Note that the empty lane is due to broken well.
PCR products were handed over to Wolfgang.
Purpose of experiment:
iLJ10: to test whether the new injection marker (Plin-44::NLS-tagBFP::let-858 3'UTR) works. I used an obvious co-injection marker (myo-2p::mCherry)
iLJ11: to test whether ceh-34p in pHW393 works as expected. Note that originally it was planned to co-inject with myo-2p::mCherry into PS7185 (GFP driver line) and later cross it into PS7203 (GCAMP driver line). I then noticed that the co-injection marker for PS7203 is also myo-2p::mCherry: syIs423 [15xUAS::?pes-10::GCaMP6s::SL2::mKate2::let-858 3'UTR + myo-2p::NLS::mCherry + 1kb DNA ladder(NEB)] V. So I decide to use rab-3p::bfp instead.
in N2 iLJ10 Volume in ul Final conc Original conc
pLJ8 Plin-44::NLS-tagBFP::let-858 3'UTR 1.3 10 156 pCFJ90 Pmyo-2::mCherry::unc-54utr 1.0 5 100 NEB 1kb ladder (0.5ug/ul) 3.4 85 500 H2O 14.3
in PS7185 iLJ11 Volume in ul Final conc Original conc
pLJ4 Pceh-34::NLS::cGAL(DBD):: cGAL(AD)::let-858 3′UTR 4.0 30 150 pMS11 pAS1-his-24::tagBFP (rab-3p) 3.1 50 324 NEB 1kb ladder (0.5ug/ul) 0.8 20 500 H2O 12.1
Note: Because I was also training Mina during my injections, I only managed to inject iLJ10, approximately 30 worms were injected.
Update: only 1 plate gives fluorescent F1s (red pharynx). 10 F1s were found and singled. I also checked the blue channel but could not find BFP in the tail. May consider to give up this as a marker.
Purpose of experiment: to miniprep some plasmid with PureLink™ HQ Mini Plasmid DNA Purification Kit for microinjection purpose.
-80 stock was streaked onto LB+Amp plates. Single colonies were picked into 3ml of LB+Amp. After O/N, 1.5ml of culture was minipreped using this kit, following the kit protocol.
pLJ4 Pceh-34::NLS::cGAL(DBD):: cGAL(AD)::let-858 3′UTR 150
pLJ5 Pceh-2::NLS::cGAL(DBD):: cGAL(AD)::let-858 3′UTR 100
pLJ6 Ptph-1::NLS::cGAL(DBD):: cGAL(AD)::let-858 3′UTR 75
pLJ7 Ptrxr-1::NLS::cGAL(DBD):: cGAL(AD)::let-858 3′UTR 93
pLJ8 Plin-44::NLS-tagBFP::let-858 3'UTR 156
pLJ9 Pins-10::NLS::cGAL(DBD):: cGAL(AD)::let-858 3′UTR 171
pLJ10 Podr-1 long::NLS::gp41-1-C-intein-cGAL(AD)::let-858 3′UTR 116
pLJ11 Pceh-34::NLS::cGAL(DBD)::gp41-1-N-intein::let-858 3′UTR 186
pMS11 pAS1-his-24::tagBFP (rab-3p) 324
260/280: all between 1.80~2.00