Difference between revisions of "Team:TPHS San Diego/Notebook"

Day 1

Miniprep bacteria with pBAD-D4 (name of the plasmid in which we will be inserting the Chitinase genes, tags, etc.) to isolate the pBAD backbone Final DNA concentration: 123.7 ng/μL

Day 2

Restriction digest using BamHI and EcoRI to check for bacterial transformation to check to make sure that the plasmid is the expected length (will also send samples for sequencing)

Wells: 1. DNA Ladder. 2. Just EcoRI 3. Just BamHI. 4. No enzyme. 5. Both enzymes

Day 3

Made KPi Buffer… (used in Chitinase Assay)

1. Prepare 800 mL of dH2O in a suitable container.
2. Add 2.405 g of K2HPO4 to the solution.
3. Add 11.73 g of KH2PO4 to the solution.
4. Add distilled water until volume is 1 L.

Day 4

Started Cloning of pBAD-GST-ChiA-FLAG construct. (Function of GST and FLAG: these are protein tags (onto chitinase) to purify and detect chitinase respectively) Did the restriction digest portion, will do gel purification, ligation, and plating tomorrow For protocol click here

Day 5

Ran gel of restriction digest of pBAD only and did Gel Purification (very straightforward after PCR, you want only the copied DNA) of restriction digest of GST/gBlock as we want to preserve the amount of DNA gBlock that we have and will lose less sample via PCR purification. Final concentrations: pBAD: 22 ng/μL gBlock: 10.6 ng/μL Did DNA ligation and plated BL21 competent cells cloned with GST-ChiA full construct (complete protocol is linked in yesterday’s log)

Day 6

Selected 10 colonies from Vector+Insert plate and put in 4 mL of LB+Ampicillin (LB is nutrients for bacterial growth, Ampicillin assists selection of transformed bacteria) media. Incubate in 37 ºC shaker for 24 hrs. Also, did restriction digest and gel on pBAD-D4 vector using MluI and HindIII to check and make sure the enzymes are cutting properly. (If DNA length match expected length, then enzymes are working properly) There is a chance we will have to do ligation and stuff again because there aren’t that many colonies. We will most likely use primers to enhance the gBlock/insert DNA and then try again. Depending on how these colonies turn out after we sequence them.

Vector+Insert colony

Day 7

Did Miniprep of the 10 bacteria colonies that we selected from Wednesday. Used NanoDrop machine to find DNA concentrations of all 10 samples.

Then we chose a specific restriction enzyme to cut both the original pBAD-D4 plasmid and our constructed plasmid such that we can distinguish between the two based on the length of their base pairs and the number of cuts that are made. We chose EcoRI-HF. This is in order to check that the plasmids that we are using are what we think they actually are. We want to have 250 ng per restriction digest reaction. Add 2 μL of buffer, 1 μL of enzyme, and fill to 20 μL with water. (Always add enzyme last). Then incubate at 37 ºC for 30 mins-1hr.

Make a 1.0% agarose gel with 150 mL 1x TAE buffer and 15 μL of Ethidium Bromide in a medium sized gel frame. Load 10 μL of DNA Ladder and put 4 μL of 6x loading dye into each sample.Run the gel at 175 V for 45 mins-1 hr.

Here is a picture of the gel simulated on a computer
program called SnapGene.

Here is a picture of the actual gel.

Wells 3-12 were extracted from bacterial colonies that all have the same DNA inserted. BUT! When we run a gel or a restriction digest in which all the samples were cut with ECORI the results are not the same. For some samples it is probably due to the enzyme not cutting the DNA, but for the rest it is unknown. We will have to do further testing to figure out what the issue is.

Resolved: The issue was likely caused by the incompatibility of the enzymes in the buffer and poor primer design. When performing PCR, it is important to add restriction sites to the primers so that the product would include the restriction site: this was not done originally. Also, the enzymes used to perform the restriction digest, HindIII-HF and MluI, were not compatible in the same buffer. The HF stands for High Fidelity, indicating that the enzyme has reduced star activity, or reduced tendency to lose specificity, and can be used in a wider range of buffers.

Day 8

Did restriction digest on pBAD-D4 vector using HindIII-HF and MluI-HF. Let incubate overnight.

Day 9

Did PCR on GST-ChiA with MluI forward primers and HindIII reverse primers. (these primers attach restriction sites onto the insert along with a buffer region in case of exonuclease action)

 	     PCR cycle: 66ºC  10 seconds (phusion)        72ºC 1.5 mins

             20 cycles

  	     50 μL DNA+6 μL CutSmart Buffer+2 μL HindIII-HF+ 2 μL MluI-HF

	     Incubate at 37ºC ~3-4hrs

 	     GST-ChiA: 60 μL = 12 μL 6x loading dye (36 μL into each well)

	     pBAD: 50.4 μL = 10 μL 6x loading dye (30 μL into each well)

             1.  pBAD extract from 3,772 bp

             GST-ChiA extract from ~2.7 kb

Day 10

Also made 5 g of colloidal chitin.
Redid PCR reaction for GST-ChiA. Ran Gel. Cut and purified ~2.7 kb

Used Qubits to find DNA concentrations.

 	     pBAD	  23.0 ng/μL
	     GST-ChiA	  39.2 ng/μL

Day 11

Transformation attempt 2

Results with Chitinase+Chitin incubation period of 2.5 hrs

We made colloids (a type of mixture between a solution and suspension, meaning that particles are small but larger than those in a solution) of chitin, which we used in a Chitinase Assay to test the viability of using the assay for the experiment. For the assay, the Chitinase concentration was diluted in a series while the amount of chitin colloid was kept constant.


This is how the assay works:vv

1. When chitinase digests chitin, chito-oligosaccharides are generated as a product.
2. This is digested using ChitO, generating hydrogen peroxide.
3. HRP uses the hydrogen peroxide to convert AAP and DCHBS into a pink compound, which allows us to visually detect the presence of chitinase.
4. To quantify the amount of chitinase present, a spectrophotometer will be used to detect the absorbance: higher means more concentrated. (absorbance = extinction coefficient * cuvette length * concentration) (chem stuff).

Day 12

Mini-prep colonies that we picked yesterday. Ran a gel and sent the purified DNA for sequencing. Selected 9 colonies:

Ran gel using EcoRI, the expected results are the same as above. The first well is 1kb DNA ladder, wells 2-10 are GST-ChiA, and the last well is pBAD-D4 vector cut with EcoRI as a control.vv

This gel is just to check that he plasmid was transformed correctly as we know the restriction sites for the cloned construct. Based on this gel, 7/9 of the clones successfully received the plasmid and we can use all of those clones (except clone 3 and 7) in experimentation.

Chitinase Assay

Assay (take.1)	Assay (supernatant only)	Results (take.1)	Results (supernatant only)
t

Based on these results, it shows that the assay protocol that we chose is sensitive enough. We performed this assay with commercially purchased chitinase in order to test the sensitivity of the assay protocol that we chose. Based on these results it seems that our protocol could be usable, however we would need to filter the colloidal chitin somehow so that there is less chunks floating around in the solution. Considering, that we now have a potent strain of GST-ChiA, we can soon use this DNA with the assay that we have to identify chitinase activity.

Day 13

The colloidal chitinase particles used in the previous assay test were too large. Redid the assay test with smaller particles. We modified the protocol based on cross references from several other colloidal chitin protocol and decided to increase the volume and chitin to HCl ratio to 1:10. We also increased the incubation period for the 12M HCl and Chitin to 2.5 hrs instead of the original 1 hr. We covered the beaker with foil as it stirred but the vapor form the acid degraded the foil and caused flakes to fall into the foil. We had to scrap it and will remake tomorrow. Also transformed BL21 Cells with complete plasmid and plate 50 μL to LB-AMP plates. Incubate at 37C overnight. Will select colonies and put in shaker O/N.

Day 14

Redid colloidal chitin, select colonies and put in 4 mL LB-AMP and put in shaking incubator (37C O/N)… prepared 10 aliquots: 4 tubes with 2 mL of LB per for clone 1 and 9 to be used for arabinose induction and WB. 2 tubes of 4 mL of LB for miniprep.

Day 15

Performed Arabinose Induction:

1. Take 100 uL of cell culture (from yesterday) and put in 10 mL of LB-AMP and put in shaking incubator for 2 hrs
2. Perform serial dilution of Arabinose

    	     a. 20% to 2% to 0.2% and do one with 0% (just H2O)

   
   
3. Add 100 uL of each dilution to one tube samples (there are 4 dilutions and 4 tubes per clone. Do this for each clone)
4. Grow at shaking incubator for 4 hrs
5. Take 1 mL of each sample, spin at max speed for 1 minute, and aspirate supernatant

Prepared samples for western blot (SDS page): Gel electrophoresis for proteins

1. Centrifuge samples and aspirate supernatant
2. Resuspend pellet in 100 uL SDS page buffer.
3. Boil 5 min and centrifuge
4. Load 12 uL of protein ladder into the first well and 10 uL of each sample and a negative
   control into the other wells.
5. Record position of samples.
6. Adjust to desired voltage and run for 1 hr

In this experimentvv, we are checking for the expression of chitinase. After the proteins are separated by molecular weight, we will transfer them onto a different membrane that will be stained with two antibodies: one to bind to chitinase, the other to help us visualize its presence by binding to the first antibody. The second antibody has an HRP (horseradish peroxidase) tag which will produce light when reacted with luminol and hydrogen peroxide. We will capture this light on a film and develop the film to get a better image. This is possible because the FLAG tag, which is included in our DNA construct, attaches itself onto chitinase and is recognized by the first antibody (called anti-FLAG) with high specificity. The second antibody recognizes the first antibody and “Brings” the HRP.

We left the membrane in blocking buffer at 4C O/N
For a step by step protocol, click here (heads up this is a more general western blot protocol and we are using different antibodies)

 	     Clone 1: Seems okay will need to sequence further to double check
             Clone 9: There is a frameshift early in the GST sequence so the protein will not be expressed properly. (For now will use as
                  negative control in western blot?)

Day 16

Finishing Western Blot:vv

        Primary Antibody: Mouse αFLAG M2
             4 mL Blocking Buffer (milk) + 1.25 λ of αFLAG (1:4000)
             (Blocking: a nonspecific antibody such as those found in milk will bind randomly, “blocking” the primary antibody and secondary antibody
                from binding to a wrong target, increasing specificity, decreasing the background noise in the film).
             Add to membrane (after draining milk) and leave on shaking @ RT for 1 hr
             Wash with TBSTE buffer 2-3 times
             Secondary Antibody: Goat derived mouse antibody? Has HRP
             10 mL blocking buffer + 1 uL of GMHRP and incubate on shaker for 1 hr
             Wash with TBSTE buffer 3 times (five minutes on shaker each time)
             Drip off excess and add 1 mL of buffer and luminol
             Prepare cassette and drip off excess before putting in membrane and take the picture. 
             Expose for 1 second, 5 seconds, 10 seconds, and 30 seconds.

Results:

(photo is 1 second exposure and no red light) Well 1-4 is for clone 1 with serial dilution of arabinose. Well 5-8 is the same for clone 9. Expected size of protein= 84.99 Dka 1 second was the optimal exposure time since it had the least amount of background disturbance.

Conclusion

We did the serial dilution of arabinose in order to determine the concentration that we would need to induce protein expression. Based on these results, 0.2% is too little but we can get induction with 2% arabinose. Well 1 had 20% arabinose, which also induced expression, but it is better to use the least concentration that is still viable. After sequencing, we discovered that clone 9 had a frameshift mutation. Thus, there is not much protein expression to be expected in this Western Blot, as seen in wells 5-8. Once we purify the protein, we can further decide if 2% arabinose is too much or too little concentration (if the purification process does not go well). Otherwise, we can continue with purification and the experiment. We also redid the chitinase assay with the colloidal chitin that we remade to avoid undissolved particles that could affect the spectrophotometer reading.

Day 17

Previously made chitin agar plates (with ampicillin and arabinose) plated GST-ChiA-FLAG construct on one plate and pBAD-D4 on another plate as a control

left:GST-ChiA. right:pBAD

As you can see there are distinct halos around the GST-ChiA colonies showing chitin degradation. We will later repeat this process using a recipe that includes calcofluor white which is a dye that will enhance the image and make it easier for us to see the chitin degradation. We also groved two samples of GST-ChiA in 20 mL of LB-Amp media O/N.

Day 18

Started protocol for GST Protein purification. Click here for full protocol.
Transferred bacterial cultures to larger aliquots of LB, induced protein expression with arabinose, and spun down the cells and saving the pellet. Also, poured LB and Chitin agar plates as well as plated transform bacteria with GST-ChiA-FLAG and GST control on 5%, 10% LB and Chitin
Plates incubating overnight at 37C. Miniprep samples of GST-ChiA and GST. (DNA concentrations- GST-ChiA: GST:)

Day 19

An issue: the bacterial cultures, for the protein purification that was supposed to be spun down for 30 minutes and then stored at -80C was accidently left in the centrifuge at 4C. Still continued with purification protocol and also prepared another round of bacterial cultures to repeat the process just in case. Up until adding the beads

PCR Tubes

 	     Clone 7
             Clone 7+ MnCl2
             Clone 10
             Clone 10+ MnCl2

PCR Reaction mix:
28 uL H2O
5 uL 10x Buffer
5 uL 10 mM dNTP
5 uL of 10 mM Primer mix (either clone 7 or clone 10)
5 uL of DNA Template (200 pg/uL)*
1 uL 25 mM MnCl2** (for non MnCl2 samples just add 1 more uL of H2O)
1 uL of Taq

*Template Stock= 145.8 ng/uL

 	     +  1uL stock and 99 uL H2O (1:100 dilution)Clone 7
                New Concentration= 1.458 ng/uL
             +  10 uL of diluted stock and 62.9 uL H2O
                Final Concentration= 200 pg/uL

 	     +  25 uL MnCl2 and 975 uL H2O
                Final concentration= 25 mM

Day 20

Ran Gel for PCR Reaction, there was no bands ㅠ-ㅠ… so redid PCR reaction. Reaction mix is the same as Thursday. PCR conditions were different.
Dilutions:
*Template Stock= 123.7 ng/uL

 	     +  1uL stock and 99 uL H2O (1:100 dilution)
                New concentration= 1.237 ng/uL
             +  10 uL of diluted stock and 61.9 uL H2O
                Final Concentration= 200 pg/uL

 	     +  25 uL MnCl2 and 975 uL H2O
                Final concentration= 25 mM

Plate w/o calcofluor white (left is ChiA, right is GST only control)	5% Chitin/LB with calcofluor white (left is ChiA, right is GST only control)	10% chitin/LB with calcofluor white (left is ChiA, right is GST only control)

Conclusion

The plates that did not have calcofluor white showed more chitin degradation and it was easier to see the degradation, this could be because the dye decreases enzymatic activity or the makes the bacteria less efficient. In any case, it will be better to use the plates without calcofluor white dye for chitinase plate assay.
The plates that did not have calcofluor white showed more chitin degradation and it was easier to see the degradation, this could be because the dye decreases enzymatic activity or the makes the bacteria less efficient. In any case, it will be better to use the plates without calcofluor white dye for chitinase plate assay.

 	     Resuspended beads and washed 200 uL with 1 mL of TKET…
                  Put 200 uL and 1 mL of TKET into eppendorf tube
                  Do a quick spin
                  Put the tube on a magnet rack
                  Remove supernatant
                  Repeat above for second wash
             Spun lysate down at 13,000 rpm for 15 mins save 50 uL of supernatant (total lysate)
             Continued to follow Protein Purification protocol
             Did Bradford assay as a quick check and there were no proteins… Did bacterial culture again. Will do protein purification again tomorrow.

Day 21

Protein Purification with the second round of colonies that we prepared. Froze all of total lysate sample (will continue process next time).
Ran a gel for PCR reaction from Monday. Still no bands…

Day 22

Redid PCR reaction according to this (take 3) We have bands!! Here are the results

DNA Ladder. Well 1: C7. Well 2: C7 with MnCl2. Well 3: C10. Well 4: C10 with MnCl2
Also finished Protein Purification still need to do Bradford assay to double check.

Day 23

Did western blot also did a quick version of the Bradford Assay.
No protein…
Will redo purification and modify the protocol