iGEM 2016 — Team IIT_Delhi

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# Team:IIT Delhi/Notebook

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  + [★ Motivation](https://2016.igem.org/Team%3AIIT_Delhi/Motivation)
  + [★ Oscillator Circuits](https://2016.igem.org/Team%3AIIT_Delhi/Cicuits)
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* RESULTS
  + [Cloning](https://2016.igem.org/Team%3AIIT_Delhi/Cloning)
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* [REFERENCES](https://2016.igem.org/Team%3AIIT_Delhi/References)

#

####

# 30th may to 7th june

Discussion on our project and the proposal sent to iBEC (Indian biological engineering competition).

## 31/05/16

Transformation of 4 plasmids using electroporation (Only 2microL of each part was used):

1. 2H6 - BBa\_R0062 - PLux
2. 5D7 - BBa\_K1399002 - RFP
3. 2L4 - BBa\_C0062 - LuxR
4. 2J4 - BBa\_C0061 - LuxI

Due to insufficient cell growth on 2 of the 4 plates transformed only 2 will be inoculated and the other 2 have to be redone.

This implies inadequate amount of plasmids were transformed.
For further processes amount of plasmid used would be around 5-6 microL

Computation:
Modelling of the Danino oscillator for a single cell.

## 02/06/16

Plasmid isolation of the 2 cell cultures.

## 04/06/16

Transformation using TSS protocol of '2 new plasmids' and '2 earlier used plasmids (insufficient cell growth)' respectively:

1. 2L4 - BBa\_C0062 - LuxR
2. 2J4 - BBa\_C0061 - LuxI
3. 2H4 - BBa\_C0060 - aiiA with degradation tag
4. 2B4 - BBa\_C0051 - lambda cI

## 05/06/16

Computation:
Discussion on Second order partial derivatives in delay differential equations.

Lab:
All 4 plates show colonies, although on one of the plates (aiiA with degradation tag part), one of the colonies does not look like a typical E.coli colony, and is transparent instead of being opaque. Anyway, we inoculated colonies from all 4 plates into LB containing tubes.
Hopefully all 4 will show growth tomorrow. That would be confirmation enough whether the colony is proper or not.
Also, we found the P+RBS (constitutive) from last year. We’ll transform that tomorrow to save it and then use it for cloning in our project.
We have also isolated a transcriptional terminator for our parts, which we will pick up from the kits tomorrow and use.

## 06/06/16

Transformation of 4 new plasmids:
1.* 2L6 - BBa\_R0065 - lambda cI and luxR
2.* 3F3 - Bba\_B0015 - Terminator
3.* P+RBS - J23119 and RBS
4.* 5H1 - BBa\_B0030 - RBS

5.

# From 8th june to 15th june

## 08/06/16

Inoculation of 3 cell colonies having the following gene plasmids:1. 2L6 - BBa\_R0065 - lambda cI and luxR promotor
2. 3F3 - Bba\_B0015 - Terminator
3. P+RBS - J23119 and RBS

The culture with the 4th plasmid didn't have sufficient growth. It has to be redone.

## 09/06/16

Plasmid isolation of the following genes:

1. 2L6 - BBa\_R0065 - λcI and luxR promotor
2. 3F3 - Bba\_B0015 - Terminator
3. P+RBS - J23119 and RBS

## 10/06/16

Gel run-

1% gel (Agarose in TAE)

Ethidium Bromide as staining dye for UV visibility

3 uL plasmid + 0.6 uL loading buffer (glycerol)

Plasmid + Backbone = Total size
1st row:
Ladder (1kb)* 5D7 - BBa\_K1399002 - RFP
* 714 bp + 2070 bp = 2784 bp
2B4 - BBa\_C0051 - lambda cI * 775 bp + 2070 bp = 2845 bp
* 3F3 - Bba\_B0015 - Terminator
* 129 bp + 2070 bp = 2199 bp
* 2H6 - BBa\_R0062 - PLuxR
* 55 bp + 2070 bp = 2125 bp
* 2H4 - BBa\_C0060 - aiiA
* 814 bp + 2070 bp = 2884 bp
* 2L6 - BBa\_R0065 - P lux-λ
* 97 bp + 2070 bp = 2167 bp
* 2J4 - BBa\_C0061 - LuxI
* 643 bp + 2070 bp = 2713 bp
2nd row:* 2L4 - BBa\_C0062 - LuxR
* 781 bp + 2070 bp = 2851 bp
* Bba\_K880005 - J23119 and RBS
* 55 bp + 2070 bp = 2125 bp
* PSB1C3 (Backbone) size: 2070 bp

All the plasmids except double terminator and LuxI showed results. This maybe because the colonies obtained of the double terminator were of green colour and the concentration of LuxI plasmid was too low.

Digestion of all the plasmids was done except the double terminator.

## 16/06/16

Transformation of the following plasmids:

1. Strong RBS BBa\_B0030 PSB1C3 Plasmid Present- Double Terminator BBa\_B0015 PSB1C3 2015 Kit Plate 3
   - LuxR gene BBa\_C0062 PSB1C3 2015 Kit plate 2
   - Double Terminator BBa\_B0015 PSB1C3 2014 Kit Plate 3
   - LuxR gene BBa\_C0062 PSB1A2 2013 Kit Plate 5
   Isolation of the 4 plasmids with their concentrations and absorbance:
   Gene Concentration A260/230 A260/280
   Plux 108.2 ng/uL 1.95 2.24
   Heme 120.6 ng/uL 1.90 2.06
   CCas Ccar 165.8 ng/uL 1.94 2.28
   Pcpcg2 286.4 ng/uL 1.92 2.22
   LuxI was also reisolated with new concentration 369 ng/uL.

   ## 18/06/16

   Plates of the transformed cell culture have the following growth:
   * Strong RBS: 1000 colonies (lawn)
   * 5O4 LuxR(2013): No growth as expected (Ampicillin resistance)
   * 2L4 LuxR(2105): 20 colonies
   * 3F3 Double Term: 100 colonies, slightly better than last time (not green)
   * 3F3 Double Term: Colonies there but medium ruptured

   ## 20/06/16

   Important discussion on the project.

   The process of producing a cloned gene in a cell culture starting from the kit plates provided by iGEM was discussed in detail. Every possible limitation and benefit of the processes came up during the discussion. This gave us a clearer view of how and why theses steps were performed and also how to overcome a limitation or problem that was encountered in them.

   Then we discussed our progress till now and planned our tasks for the next few days.

   ## 21/06/16

   Digestion of PSB1K3 and PSB1A3
   Gel run:
   •* Gel contents: 1% Agarose in TAE(100mL) and Ethidium Bromide(7.5uL)
     •* Loading dye 5X: 1uL dye + 4uL plasmid
       •* PLux 55
       •* PLux-λ 97
       •* LuxI 643
       •* aiiA 814
       •* RFP 714
       •* P+RBS 55
       •* Ladder 1kb
       •* RBS 15
       •* RBS 15
       •* Terminator 129
       •* Terminator 129
       •* Terminator 129
       •* RFP 7141)Promoter very faint band
   2)backbone very faint( loading problem)

   ## 22/06/16

   Calculation of amounts of plasmid content.
   Ligation of 12 plasmids in pairs as follows:
   PLux + RBS
   LuxI + T
   J23104&RBS + RFP
   Plux-λ + RBS
   RFP + T
   aiiA + T

   ## 23/04/16

   Made chloramphenicol
   TAE(50X) and MilliQ prepared
   Autoclaved apparatus and LA
   Transformation of the 6 cloned plasmids using TSS method.
   10 Amp, 11 Kana, 12 Cat plates prepared.
   Spreading of the 6 cultures.
   Plates kept in shaker at 37C for 16 hrs.

   ## 24/06/16

   3 colonies of each of the 6 plates inoculated to 18 tubes.
   Streaking of 3 samples (Kanamycin) to neomycin plates to check
   if Kanamycin is working fine or not.
   50 colonies of J23104 + RFP streaked for confirmation of RFP.

   ## 25/06/16

   Plasmid isolation from the 18 tubes.
   After the isolation, 2 vials were removed due to loss of plasmid content
   from them, namely aiiA + T1 (7th vial) and aiiA + T3 (18th vial),

   ## 26/06/16

   Digestion of the 6 clones from the 16 tubes.
   Gel run was done as follows:
   Gel run 3% for 15 min:
   1. Empty
   2. PLux + RBS-1 (11)
   3. PLux-λ + RBS-1 (12)
   4. 100 bp ladder
   5. PLux-λ + RBS-2 (14)
   6. PLux-λ + RBS-3 (15)
   7. 50 bp ladder
   8. PLux + RBS-2 (16)
   9. PLux + RBS-3 (17)
   10. Empty
   3% final
   • Loaded the samples (around 15uL)
   • While loading, sample 12 (PLux-•

   ## 27/06/16

   Plasmid isolation of new aiiA + T
   Digestion of aiiA + T and LuxR

   Gel run 1%:
   1. LuxR c(undigested)
   2. LuxR
   3. Ladder
   4. aiiA + T
   5. aiiA + T c(aiiA)

   Gel run 3%
   3. T
   4. ladder
   5. T control

   ## 29/06/16

   Plasmid isolation of the following 8 genes:
   LuxI
   Terminator(x2)
   PLux + RBS
   PLux-λ + RBS
   RFP + T
   J23104-RBS
   Gel run 1%
   • PLux + RBS and PLux-λ + RBS confirmed
   • LuxI showing a band of 9-10 kb
   • Terminator band not visible due to low concentration

   Inoculation of 3 new colonies each of Terminator and LuxI

   ## 30/06/16

   Plasmid Isolation of T and LuxI

   Gel run 1%
   • Terminator isolated from the kit was confirmed.
   • All 3 LuxI gave a band of 8-10 kb. So LuxI is still unconfirmed.

   Inoculation of 4 colonies of aiiA (3 transparent + 1 white)
   Transformation of 2016 parts

   ## 1/07/16

   Culture of aiiA picked up from the white colony was of green colour.
   So aiiA was isolated from the remaining 3 cultures in a single falcon.

   Digestion of T, aiiA and PSB1A3

   Gel run 1%
   1. T digested
   2. T control
   3. Ladder
   4. aiiA digested
   5. aiiA control
   6. PSB1A3

   All 3 confirmed although band of aiiA was extremely faint.

   Ligation: aiiA + T

   Inoculation of all 2016 parts:
   LuxI
   RBS + LuxI + T
   LuxR
   aiiA
   RBS + aiiA + T
   PLux-λ + RBS
   Terminator

   ## 2/07/16

   Isolation of 2016 parts(7)

   Gel run 1% of all the 2016 parts
   • LuxI and Lux R showing band of 9-10 kb (unconfirmed)
   • RBS + LuxI + T , RBS + aiiA + T , aiiA and PLux-λ + RBS confirmed!

   Transformation: aiiA + T

   ## 3/07/16

   Digestion of RBS + LuxI + T , RBS + aiiA + T , PLux and PLux-λ

   ## 4/07/16

   Plasmid isolation of 2 PLux: small and big colonies

   Nanodrop:
   PLux small - 243 and 2.00 , 2.10 ratios
   PLux big - 311 and 1.99 , 2.19 ratios

   Digestion of RBS + LuxI + T , RBS + aiiA + T , PLux(x2) and PLux-λ

   Gel run 1%

   ## 2.07.2016

   • Firstly, we did plasmid isolation ( 2 manually, 1kit) which was followed by Gel Run.
   • Then we set up the digestion for 3 hours and then did the gel run for the digested plasmids.
   • At the end of the day, we did ligation of the digested products.

   ### Plasmid Isolation

   Plasmid |Manual |Kit(Concentration)
   PL+RBS| 0| 1(210)
   T(old) |2 |1(700)
   RFP |2 |1(222)
   RFP+T |2 |1(153)

   ### Gel Run

   1.PL+RBS
   2.T(K)
   3.T(M1)
   4.T(M2)
   5.Ladder
   6.RFP+T(K)
   7..RFP+T(M1)
   8..RFP+T(M2)
   9.RFP(K)
   10.RFP(M1)
   11.RFP(M2)
   PL+RBS: confirmed
   T(old): confirmed
   RFP: confirmed
   Pl=RFP+T: not confirmed

   ### Digestion

   The total reaction volume was 20µl.
   Plasmid Amount added in digestion (µl)
   PL+RBS (E/S) 7.5
   T(X/P) 2.3
   T(E/S) 2.3
   RFP (E/S) 7.5
   RFP (X/P) 7.5
   RFP+T (X/P) 10
   J23104+RBS (X/P) 7.5

   ### Gel Run

   Well 1 Confirmed.
   Well 2 Confirmed.
   3 Confirmed.
   4 Confirmed.
   5 Confirmed.
   6 Saw its backbone.
   7 ladder.
   8 Saw its backbone.
   9 Saw its backbone.
   10 Saw its backbone.
   11 Confirmed.
   12 Confirmed.
   13 Confirmed.

   ### Ligation

   Total reaction volume was 30 µl and vector added was 2 µl.
   Ligation Plasmid Volume added.
   (RFP) + (T) [kana] 3.2+2.6.
   (PL+RBS)+(RFP+T) [chloram] Not done because we did not have chloramphenicol.
   (PL+RBS)+RFP [amp] 2.8+3.2.
   T+(J23104+RBS) [amp] 2.6+2.3.

   ## 4.07.2016

   ### Digestions

   Total reaction volume is 20µl.
   Plasmid |Amount added in digestion (µl) .
   PL+RBS (E/S) |3.5.
   T(X/P) |2.3.
   T(E/S) |2.3.
   Chloramphenicol(E/P)| 10.
   Ampecillin(E/P) |10.

   ### LIGATION

   Plamids |Volume added.
   (PL+RBS)+(RFP+T) [chloram] | 2.8+3.6.

   ## 5.07.2016

   ### TRANFORMATION

   Terminator+ (J23104 +RBS) 15 µL AMP
   RFP+Terminator 15 µL KANA
   (PLuxL+rbs)+RFP 15 µL AMP
   Aiia+Terminator 15 µL AMP
   (PLuxL+RBS)+(RFP+Terminator) 15 µL CAT
   (Promoter + RBS+LuxI)+Terminator 15 µL AMP
   (Promoter + LuxR+ Aiia)+Terminator 15 µL AMP
   17d AMP
   17p AMP
   We saw lawns in all the amp plates. There is contamination in the plates. It could either be yeast .

   # WEEK – 7

   • Transformation – TSS
   • RFP(E/S-PM-Eluted)+T+psb1k3==>Clone
   • T+psb1k3 ,transformation completed
   • Transformation results
   • We observed 2 colonies in clone.RFP(E/S-PM-Eluted)+T+psb1k3.RTK3.1
   • No colonies were in negative control (without RFP)

   • plasmid isolation of RTK3.1
   • Observed late growth in RTK3.1-2
   • Digestion with ecor1
   • PRL(part)

   • Plasmid isolation:-
   • Vigoruous lysis==> saw genomic DNA too
   • PCR

   • PRRT(part)

   ## 24/7

   • Plasmid isolation:-
   • Vigoruous lysis==> saw genomic DNA too
   • PCR

   • Gel run results:-
   • Pst1 not working properly- PRL lot of undigested is present.
   • PluxL+RBS digested is perfect. Saw a band below 250bp around 113bp.
   • A lot of single digested is left after pst1 digestion. Xba1 not working properly after Pst1.

   • Ligation of RFP+T
   • Ligation of RFP+T(psb1c3)+psb1k3
   • Digestion of psb1K3

   • PCR by shashank
   • Reaction of (R+luxR+T)
   • Remarks :- saw very little on gel.

   • Digestion of psB1A3 and psB1T3
   • Psb1A3
   • Plasmid isolation through kit
   • Gel of plasmid = 3ul (very intense bands)l
   • Digestion of j23100

   # 6/8/16 to 14/8/16

   PLuxL+RBS
   **Plasmid isolation**
   Nano drop reading ==> 174.3ng/ul
   260/280=1.99
   260/230=2.09
   Gel run of plasmid
   Digestion = 50ul
   Plasmid = 20ul = 3.4ug
   Enzymes -E/S=1ul glycerol = 1%
   DNA conc. = 70ng/ul (standard NEB = 50ng/ul)
   RFP+T
   **Plasmid isolation**
   Nano drop reading ==>
   260/280
   260/230

   280.6ng/ul
   1.94
   2.23

   **Gel eluted**
   Psb1k3

   **Ligation**
   PluxL
   1ul = 31mol
   Ratios:2

   Rfp+t
   3ul = 17.6mol
   1

   1k3
   1ul = 13.6 mol
   1

   PluxL
   1ul = 31mol
   Rfp+t
   3ul = 17.6mol
   1k3
   1ul = 13.6 mol

   # 22ND August to 29th August

   Plux + RLIT
   Gel
   * Gel Elute RLIT
   * Nano Drop
   * Gel Check RLIT
   * Ligations
   PλRAT(220ng/ul) + PRRT(224ng/ul)
   * Plasmid isolation - 1600
   * DigestionRLuRTP
   Transformation()
   J23100 promoter plasmid/Disgested ask? And then verify
   Digestion 1k3
   **PLRAT 220ng/ul**

   Digestion = 50ul
   Divide in 2 and add 0.5ul of enzymes.
   PLRAT E/S = 25ul (66ng/ul)
   PLRAT X/P = 25ul (66ng/ul)
   PRRT 220ng/ul

   Digestion
   Divide in 2 and add 0.5ul of enzymes.

   PRRT E/S=25ul (66ng/ul)
   PRRT X/P = 25ul (66ng/ul)

   1.Ligation of plux+(RLIT)+1K3(iGEM) (4:2:1)
   2.Ligation of plux+(RLIT)+1K3(iGEM)(1:1:1)
   3.Ligation of plux+(RLIT)+1k3(iGEM) (8:2:1)
   4.Ligation of plux+1k3(iGEM) Control (8:2:1)
   5.Ligation of plux+(RLIT)+1K3(GE) (1:1:1)
   7.Ligation of PλRAT+PRRT+1K3 (2:2:1)
   6.Ligation of PλRAT+PRRT+1K3(iGEM)(2:2:1)

   # 6/9 to 13/9

   Digestion PL-PR-Elution
   Verify LuxR
   Gel elute PL-PR==>E/S and X/P
   PL-PR band visible with distinction of digested backbone.
   Taking my history into account possible that E/S and X/P intermixed.
   After gel elution 5ul on gel - No bands visible

   Ligations
   • LuxI and LuxR
   • LuxI and PL-PR(with BB)
   • LuxI
   Strategy to characterize parts
   Work on ADDGENE PARTS.
   Ligation with pbluescript
   • LuxI with sk(+)
   • LuxR with ks(+)
   • LuxIR with RFP
   Ligation with 1C3
   • LuxI
   • LuxR
   • aiiA
   • RFP
   • LuxIR with 1C3
   Gel verification
   aiiA
   RFP
   PL-PR E-S GE
   Pl-PR X-P GE
   Ligations with 1C3
   Ligations with sk+
   L10 CONTROL

   Psl1180
   Performed Electroporation of LuxIR
   • Pellet was alright
   • Poured 12.5ul in one and 8ul in another.
   • One of them arched - not sure exactly which one.
   • Already made plates of kana were used(assuming to be working).
   • For some reason while spreading resistance came very early.
   • Have also inoculated 5ul of recovery into LB.

   Results 1st batch of transformation failed.
   Reason ==>
   • 1st time with gblocks.
   • LuxI and LuxR were verified on gel.
   • Problem with desalting possible.
   • Enzymes are not working properly on DNA.

   Ligations with 1C3l
   Digestion LuxI - Verification+ligation
   Digestion LuxR - Verification+ligation
   Digestion Aiia - Verification+ligation
   Digestion RFP - Verification+ligation
   Results: - They are where they are supposed to be and conc
   Was also what we expected.

   Desalting of 10 ligations.
   3 plates of chloramphenicol and 5 of amp.
   Need some amp plate to verify amp plate.

   Digestion of 1C3= 20ul
   Digestion of LuxI = 20ul

   # 14/9 to 21/9

   Sterilizing cuvettes
   Wednesday, September 14, 2016
   2:19 AM
   10 times MQ
   Fill with ethanol 10min
   Dump it out
   Put it in petri dish incubate in UV for 15min
   Put them in oven for 5-6 hrs or overnight
   Kshitij did an experiment to check contamination of DNAses in tank.
   In one ladder he poured 20mM of MgCl2.
   Stefan sir were right smear was because of DNAses.
   He said problem was because of tank.
   So from now put gel in 4c and use fresh TAE.

   LIGATION OF LuxIR with 1C3
   LIGATION OF LuxIR with 1C3
   LIGATION OF LuxIR with 1K3
   LIGATION OF LuxIR with 1K3

   Ligations with 1K3
   PL-PR with PRLIT
   Gel check PL-PR
   Digest Pl-PR with E/S- sequential
   Gel Check
   Ligation
   Keep some for gel elution tomorrow
   Gel Check
   • PRLIT and as control RLIT.
   PL-PR digested.

   # 22nd September to 29th September

   Make reagents for plasmid isolation - Ayush and Zode
   Autoclave of LB , MQ, and others.
   Send parts for sequencing - give work to someone - K.Vinay all the clones send everything.
   Digestion of luxI, LuxR and 1C3 and 1K3.
   Gel verification
   12 colonies LuxR
    -
   Digested LuxIR

   Ligations

   LuxIR(IDT)
   L1 1:1:1 kana

   LuxI-E/S(IDT)[9ng/ul] = 1.2ul = 0.011
   LuxR-X/P (IDT)[9ng/ul] = 1.2ul = 0.011
   1K3 -E/P [12.5ng/ul] = 2ul = 0.011
   Ligase = 1ul
   Buffer = 2ul
   MQ = 12.6

   L2 3:3:1 kana
   LuxI-E/S(IDT)[9ng/ul] = 3.6ul = 0.033
   LuxR-X/P (IDT)[9ng/ul] = 3.6ul = 0.033
   1K3 -E/P [12.5ng/ul] = 2ul = 0.011
   Ligase = 1ul
   Buffer = 3ul
   MQ = 16.8ul

   L3 CAT
   PRLIT +LuxR
   PRLIT = 2ul
   LuxR = 1.2ul
   1C3 = 2ul
   Buffer = 2ul
   Ligase = 1ul
   Mq= 11.8ul

   Ligations
   PLIRT
   L4 CAT
   PRLIT E/S = 2 ul
   PLURT X/P = 2 ul
   1C3 = 2ul
   Ligase = 1ul
   Buffer = 3ul
   MQ = 20ul

   L5 CAT
   PRLIT E/S = 4ul
   PLURT X/P = 1 ul
   1C3 = 2ul
   Ligase = 1ul
   Buffer = 3ul
   MQ= 19ul

   L6 CAT
   PRLIT E/S = 2 ul
   PLURT X/P = 2 ul
   1C3 = ------
   Ligase = 1ul
   Buffer = 3ul
   MQ = 20ul

   L7 control for ligase Amp
   Pcks+ = 2ul
   Ligase = 1ul
   Buffer = 2ul
   MQ = 15ul

   PRLIT is confirmed to be at the required size.
   Innouclate PRLIT and plasmid isolation using kit.
   Gel elution plURT and PRLIT
   Autoclave 100 LB, tips, MCT's 2ml -- Ayush
   Prepare own TSS -- Angad
   Again transformation of remaining ligated mixture -- Shashank
   luxIR2 1 and 3 ki digestion with
   ask stefan for ligase 10ul

   # 28th September to 15th October

   Digest 1k3 and 1c3
   Send PRLIT for sequencing ASAP.
   Transformations tomorrow.
   1C3 and 1K3 verified on gel. Perfect picture. Important observation on gel
   PRLIT and PLuRT also verified on gel.
   Everything is set for liagtions.
   To make TSS of my own

   Preparing LA plates by putting 2 flasks of 300ml failed
   Somehow heat was not enough for their boiling but enough for the charring.
   It took much longer to boil them and in one them because of improper mixing and late boiling (which ultimately helps in mixing) led to charring of chunk of mass.

   Ligation of PRLIT and PLuRT
   Digestion of LIR(E/S) and PLPR(X/P)
   Desalting of LIR and PL-PR
   Heat inactivate
   Digest with Sma1
   Gel Run
   Gel elution
   Ligation of LIR and PLPR with 1K3 and 1C3.
   Single digestion of same plasmids with pst1 of LIR - 1 and 4 - to verify
   Digestion of false clones to check what they really are.
   Digestion = 40ul of LIR
   Digestion = 40ul of PL-PR
   Tranformations
   PcstA+RBS+C1
   pBAD+RBS+CI
   LIR
   PL-PR
   Pacyc184

   There is turbidity in our buffer2.1.
   All this time problems might be because of bufffer2.1
   Digestion = 20ul 0f 1C3 and 1K3

   5.Digestion of LIR = 40ul
   There was no smear in the gel.
   While eluting 1C3 some of the ladder was also taken
   6.Digestion of PL-PR = 40ul
   7.Digestion of psb3t5 -E/P= 50ul

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