May 12

Congratulations with the First Working Day!

May 13:
• Camelid's antibody p53 sequence was found
• Exploring camelid antibodies:

1) Nanobodies: Natural Single-Domain Antibodies by Serge Muyldermans, March 2013

2) Nanobody-based products as research and diagnostic tools by Thomas De Meyer et al, 2013

May 17: We recieved the reply from Dr. Fernandez lab who are ready to send the plasmid containing the zipper and HlyA sequence. Special thanks to Dr. Shipley who wrote the letter.

May 19: We had discussed the three topics, the nanobody (nb) project's protocol is ready, antimicrobial peptides (amp) and phi29 are under the process. We distributed the people from nb group to support othre projects. The new papers and nb presentation were uploaded on the google drive

June 4: We did DNA miniprep of E. coli cultures in LB medium, which was obtained from plates with transformed plates. Cells were transformed with the parts from the kit: BBa_K608002 and BBa_E1010. The tubes with miniprep products are at +4C

Concentrations of DNA measured with Nanodrop:
• Promoter: 221.3 ng/ul
• RFP: 348.2 ng/ul
• RFP from Arabinose plates: 230.2 ng/ul

June 13:

We did miniprep of DNA from sigle colony isolation (see June 11) and checked our DNA concentration on nanodrop: [BBa_I15016]=206.7ng/ul and [RFP control]=375.1ng/ul

June 16. Dear team! I am glad to inform you that we have received stab of E. coli with the plasmid containing the zipper and HlyA sequence, and a stab of E. coli with pVDL9.3 encoding hlyB and hlyD!!! A big thank to Dr. Fernandez Herrero and to Dr. Shipley!

June 19: We did pcr of Chloramphinecol plasmid backbones, and we used the following reagent mixture set up for 20ul:

See agarose gel picture for the results.

June 20: We did pcr of backbones and rfp part again, but using 3 best annealing temperatures. We have also used concentration of primers for the backbone twice as low as last time, and the bands are very clear. See the picture

June 21-22: We did transformation of e.coli with 3 part+1 control. PelB and HlyA worked well, but LigA, which was planned to be used as 75kDa protein did not work. I found out, that it requires the special KRX strain of e.coli, which we do not have. So, I took another part (Salty_VrgP) from the kit which 79kDa protein and did the transformation; also I did transformation with NahR gene, which gives 34kDa protein, we will use it in constructing "rehearsal" part of the "competence project". In addition, we did miniprep of plasmids, that we've got from Madrid and of PelB and HlyA.

June-23: we did the competent dh5a cells. The transformation was conducted The cells are competent.

July 19: We decided to check expression of VHamylase in E. coli from Madrid.

The whole protocol of what we did:
1. Inoculate a single colony of the bacteria from plate into 5ml LB with 100ng/ul Ampicillin, 33ng/ul, chloramphenicol, and 0.5mM IPTG. Shake for 12 hours at 150 rpm at 30degC
2. Centrifuge the overnight colonies at 4000g for 40 min
3. Take 4ml of the supernatant and mix with 800ul of 25% Trichloroacetic acid (TCA). Leave the samples at -20degC overnight
4. Centrifuge the samples for 1hour at 14000g at +4degC, then wash twice with acetone and let dry the pellets for 2 hours at room temperature
5. Resuspend the pellets in 100ul sample buffer (Laemmli+b-mercaptoethanol) and boil at 95C for 10min
6. Run the samples on SDS PAGE, stain with Coomasie blue and then destain for 24 hours
7. We observed the bands of size 41kDa. See the picture for the results, but it is not seen clearly on the picture, probably, because of low concentration of the protein. Note: Size of 41kDa indicates the fact that secreted protein was not dimerized because dimerized protein was expected to have the size of 84 kDa

July 19: We did restriction digest of miniprep product (GFP in plasmid backbone) using EcoRI and SpeI:

1. Digestion with SpeI: DNA - 2ul (500ng), 10xTango buffer - 2ul, Water - 15.5ul, SpeI (Fermentas) - 0.5ul. We incubated the reaction at 37C for 1.5 hour

2. Then we added 2.5ul of EcoRI buffer and 0.5ul EcoRI (Fermentas) to the initial reaction.

3. We ran the reaction mixture on the gel and observed that the digestion worked (see the picture, third well): we have got band for the plasmid backbone (2070bp) and for GFP (~700bp)

July 21: we decided to do the double digest of plasmid with EcoRI & PstI.

Total reaction mixture - 20ul: DNA miniprep product (700ng) - 2.0ul,10xBuffer SH - 2ul,EcoRI (Invitrogen) - 1ul, PstI (Sigma) - 1ul. We incubated the reaction at 37C for 1hour, then inactivated the enzymes at 65C for 15 min. The double digestion worked: we were able to see two distinct bands (for plasmid backbobe and for GFP). See the image.

July 27: we tried a new method of assembling parts, which does not require the use of restriction enzymes and ligase. The method is Circular Polymerase Extension Cloning (CPEC).

Reaction mixtures 25ul each:

a) for incorporating NahR (34kDa) protein into backbone (3338bp total): Purified PCR product of backbone (100ng) - 4ul, Purified PCR product of NahR (100ng) - 4.2ul, HF master mix - 11.75ul, DMSO - 0.75ul, dWater - 4.3ul

Thermocycler program: 30s - 98C, 10s - 98C, 30s - 55C, 50s (15s/kb) - 72C, 15 cycles, 10min - 72C

b) for incorporating INP into backbone (3000bp total), Not purified PCR product of backbone (700ng) - 1.02ul, Not purified PCR product of INP (700ng) - 1.81ul, HF master mix - 11.75ul, DMSO - 0.75ul, dWater - 9.67ul

Thermocycler program: 30s - 98C, 10s - 98C, 30s - 55C, 45s (15s/kb) - 72C, 15 cycles, 10min - 72C

Results: We observed the bands of the expected sizes on the gel, which means that the desired incorporation did occur. See the image for the results. We will transform the obtained products into E. coli tomorrow in order to confirm that the experiment worked.

July 27: Another SDS PAGE for VHamylase. This time we did two types of samples: unheated and heated. We were able to observe the sizes of proteins for both dimerized proteins and monomers. See the picture.

July29: Today we combined 2 parts together and incorporated them into backbone with CPEC: INP+NahR(34kDa)+Backbone.

1. We digested INP with SpeI and NahR with XbaI for 2 hours at 37C, then heat-killed the enzymes at 65c for 15min: DNA (directly from PCR) - 10ul, dWater - add up to 30ul, 10XRecomended Buffer - 2ul, Restriction Enzyme -1ul

2. We combined 3 genes with CPEC:

Reaction mixture (25ul):Backbone - 2ul, INP with digested SpeI site - 2ul, NahR with digested XbaI site - 2ul, High Fidelity Master - 11.75ul, DMSO - 0.75ul, dWater - 6.5ul

Program: the same as in my previous post, just the extension time was increased to 64s, due to the fact that we want to construct 4268bp long part. See the picture of the gel.

October 13

We have eventually received our genes (gp16 and Vhp53) in pUC57 vector backbone (http://www.genscript.com/app/product/downfile.html?from=1&file=filecenter/document/1400_20060331011034.JPG) and primers. We transformed Dh5alpha cells with these plasmids

October 16

The size of amplified Gp16 gene was 1056bp, while the size of nanobody construct (Vh p53 + Ig hinge + his tag + HlyA) was 1995bp