Team:Aachen/Notebook/Documentation/Methanol Single Expression


Laboratory Notebook

construct description sequencing confirmed plasmid confirmed cryo
RBS.mdh in pSB1A30 #BZBQ# #IGEM#
RBS.hps in pSB1A30 #4PAB# #4P9H#
RBS.phi in pSB1A30 #PEA6# #TFE1#
RBS.xpk in pSB1A30 #1NFT# #ONK4#

15-06-16

restrictions:

  1. B0015 in pSB1C3 #TK6C# with XbaI and PstI
  2. hps in pSB1C3 #H4M9# with EcoRI and SpeI
  3. phi in pSB1C3 #ANNA# with EcoRI and SpeI


calculation:

components Volumes for (1.) B0015 [µl] Volumes for (2.) hps [µl] Volumes for (3.) phi [µl]
ddH2O 14.7 15.2 15.5
10x NEBuffer 2.1 2.0 2.0 2.0
Enzyme I 0.4 (XbaI) 0.4 (EcoRI) 0.4 (EcoRI)
Enzyme II 0.4 (PstI) 0.4 (SpeI) 0.4 (SpeI)
DNA 2.5 (#TK6C#) 2.0 (#H4M9#) 1.7 (#ANNA#)


  • restriction products stored in Falcon #TUSS#

ligation:

  • (A) (1.) B0015 digest and (2.) hps digest into pSB1K30 digest (#SZKD#)
  • (B) (1.) B0015 digest and (3.) phi digest into pSB1K30 digest (#SZKD#)
components Volumes for (A) [µl] Volumes for (B) [µl]
ddH2O 7 7
10x T4 Ligase Buffer 2 2
upstream Part Digest 4 (B0015 digest) 4 (B0015 digest)
downstream Part Digest 4 (Hps digest) 4 (Phi digest)
pSB1K30 digest #SZKD# 2 2
T4 DNA Ligase 1 1


  • ligation products stored in falcon #TUSS#
  • heatshock transformation in DH5α, plate on LB + K

15-06-17

  • because backbone is probably not well digested (and because we have all four genes right now!!), we make the restriction and ligation again

restriction

  • xpk in pSB1C3 (#KFEY#) with EcoRI and SpeI (X)
  • mdh in pSB1C3 (#9PBD#) with EcoRI and SpeI (M)
components Volumes for Xpk (X) [µl] Volumes for mdh (M) [µl]
ddH2O 16.5 15.4
10x NEBuffer 2.1 2.0 2.0
Enzyme I (EcoRI) 0.4 0.4
Enzyme II (SpeI) 0.4 0.4
DNA 0.7 (#KFEY#) 1.8 (#9PBD#)
  • xpk restictions: tube X
  • mdh restrictions: tube M

ligation

  • (A) B0015 digest from #OHOZ# and (2) hps digest from #TUSS# into (K30) pSB1K30 digest from #SZKD#
  • (B) B0015 digest from #OHOZ# and (3) phi digest from #TUSS# into (K30) pSB1K30 digest from #SZKD#
  • (C) B0015 digest from #OHOZ# and (X) xpk digest from #OHOZ# into (K30) pSB1K30 digest from #SZKD#
  • (D) B0015 digest from #OHOZ# and (M) mdh digest from #OHOZ# into (K30) pSB1K30 digest from #SZKD#
components Volumes for (A) [µl] Volumes for (B) [µl] Volumes for (C) [µl] Volumes for (D) [µl]
ddH2O 7 7 7 7
10x T4 Ligase Buffer 2 2 2 2
upstream Part Digest 4 (B0015 digest B0015 from #OHOZ#) 4 (B0015 digest B0015 from #OHOZ#) 4 (B0015 digest B0015 from #OHOZ#) 4 (B0015 digest B0015 from #OHOZ#)
downstream Part Digest 4 (hps digest 2 from #TUSS#) 4 ((phi digest 3 from #TUSS#) 4 (xpk digest X from #OHOZ#) 4 (mdh digest M from #OHOZ#)
pSB1K30 digest (plasmid from #SZKD#) 2 2 2 2
T4 DNA Ligase 1 1 1 1

Trafo

  • heatshock transformation in BL21

15-06-18

  • make overnight cultures and masterplates
    • 12 clones of each construct were plated on master plates. 6 overnights were made.

15-06-19

Colony PCR

gene / clones tube number expected length machine elongation time result
mdh.B0015 in pSB1K30 clones 1-12 37-48 1682 2 1'50" Negative for all clones. Observed bands at 1227 bp (RFP)
hps.B0015 in pSB1K30 clones 1-12 13-24 1160 1 1'13" Negative for all clones. Observed bands at 1227 bp (RFP)
phi.B0015 in pSB1K30 clones 1-12 1-12 1079 1 1'13" Negative for all clones. Observed bands at 1227 bp (RFP)
xpk.B0015 in pSB1K30 clones 1-12 73-84 3002 3 3'05" Negative for all clones. Observed bands at 1227 bp (RFP)
  • We picked only "red" clones, because we forgot to induce with IPTG.
  • with green light the red flourescence could be observed very well
  • we picked the few white clones and put them on a masterplate LB+K
  • next step will be a new colony PCR on monday.

15-06-22

colony PCR

  • tube numbers on masterplate
gene / clones tube number expected length machine elongation time result
mdh.B0015 in pSB1K30 clones 1-3 6-8 1682 2 1'50" bands are too short
hps.B0015 in pSB1K30 clones 1-3 1-3 1160 1 1'13" bands are too short
phi.B0015 in pSB1K30 clones 1-2 4-5 1079 1 1'13" bands are too short
xpk.B0015 in pSB1K30 clones 1-3 9-11 3002 3 4'00" bands are too short
  • restriction/ligation didn't work out as we expected.
  • problem: pSB1K30 digest never works properly
    • next step: plate cryo #8T49# to get more plasmid of pSB1K30

15-06-23

  • cut #LYZH# (pSB1A30) with EcoRI and PstI
  • cut #TK6C# (B0015 in pSB1C3) with XbaI and PstI
    • reaction volumes: 50 µl
components volumes [µl] for pSB1A30 digest volumes [µl] for B0015 digest
H2O 38.5 36.8
NEBuffer 2.1 5 5
Enzyme I 1 (EcoRI) 1 (XbaI)
Enzyme II 1 (PstI) 1 (PstI)
DNA 4.5 (#LYZH#) 6.3 (#TK6C#)
  • restrictions stored in Falcon #OHOZ#
  • gel check of digests:
digest expected bands slot on gel result
#TUSS#:2 684/2051 5 corresponding bands
#TUSS#:3 558/2051 6 corresponding bands
#OHOZ#:X 2526/2051 2 corresponding bands
#OHOZ#:M 1206/2051 1 corresponding bands
#OHOZ#:1 902/2173 3 corresponding bands
#OHOZ#:2 155/2052 4 bands of this length not visible in general on gel
  • ligate each RBS.CDS digest with the B0015 out of pSB1C3 into pSB1A30 digest
components (A) RBS.mdh.B0015 in pSB1A30 volumes [µl] (B) RBS.hps.B0015 in pSB1A30 volumes [µl] (C) RBS.phi.B0015 in pSB1A30 volumes [µl] (D) RBS.xpk.B0015 in pSB1A30 volumes [µl]
H2O 7 7 7 7
T4 Ligase Buffer 2 2 2 2
upstream part digest 4 (#OHOZ#:M) 4 (#TUSS#:2) 4 (#TUSS#:3) 4 (#OHOZ#:X)
downstream Part digest (#OHOZ#:2) 4 4 4 4
backbone digest (#OHOZ#:1) 2 2 2 2
T4 Ligase 1 1 1 1

ligation products stored in falcon #CAJO#

  • heatshock transformation into BL21. Don't forget induction with IPTG for red/white screening!

15-06-24

  • make masterplates
  • again do heat shock transformation in BL21 cells with the ligation products (#CAJO#)
  • purify plasmid of pSB1K30. plasmid stored in: #1K30#

15-06-25

  • do colony PCR and make overnights of successful clones
  • perhaps new masterplates of transformants from 15-06-24

Colony PCR

  • with KAPA 2G Fast Ready MasterMix

Programm:

! Step Time Temp
denature 3'00 95
denature 0'15 95
anneal 0'15 58
elongate 0'25 (Machine1) / 0'45 (Machine2) 72
elongate 2'00 72
store forever 16

30 cycles!

gene / clones tube number expected length machine elongation time result
RBS.mdh.B0015 in pSB1A30 clones 1 1 1682 2 0'25 no bands
RBS.hps.B0015 in pSB1A30 clones 1-3 2, 3, 4 1160 2 0'25 no bonds
RBS.phi.B0015 in pSB1A30 clones 1-6 5, 6, 7, 8, 9, 10 1079 2 0'25 very bad bands
RBS.xpk.B0015 in pSB1A30 clones 1-3 11, 12, 13 3002 5 0'45 very bad bands

new transformants

  • make masterplates of all transformants WITH IPTG!
    • Plate 1: 26 g --> 26 µl 1000x IPTG
    • Plate 2: 30 g --> 30 µl 1000x IPTG
  • make overnight cultures of 35 clones

15-06-26

Colony PCR

gene / clones tube number expected length machine elongation time result
RBS.mdh.B0015 in pSB1A30 clones 6, 8, 10, 11 1-4 1682 2 0'25 no bands
RBS.hps.B0015 in pSB1A30 clones 3 5 1160 2 0'25 no bands
RBS.phi.B0015 in pSB1A30 clones 2, 4, 6, 7, 8, 13, 17-21 6-16 1079 2 0'25 no bands
RBS.xpk.B0015 in pSB1A30 clones 1, 7 17, 18 3002 5 0'45 no bands
  • control with red clones: mdh (tube MR), hps (tube HR), phi (tube PR), xpk (XR)

We have to think about a new strategy, as the Biobrick assembly seems not to work

15-06-29

A new beginning! Work carefully! Our new approach is to ligate the RBS.CDS constructs into a prefix cut (with EcoRI and XbaI) pSB1A3 vector containing the B0015.

In a next step we will swap the RBS.CDS.B0015 constructs into pSB1K30 backbones.

Monday and Wednesday are the days with the most workload, while tuesday and thursday give space to plan further steps

  • cut #FW6L# with EcoRI and XbaI; reaction volume: 50 µl; water: 36.3 µl; DNA: 6.7 µl
  • cut #1K30# with EcoRI and PstI; reaction volume: 50 µl; water: 37 µl; DNA: 6 µl
  • control of digests on gel
    • expected lengths:
      • BEX: 19 bp / 2273 bp (bands at expected length)
      • K30: 902 bp / 2222 bp (very weak bands)

As digest of pSB1K30 never works, we will now use pSB1A30


  • digest RBS.CDS constructs with EcoRI and PstI
    • RBS.mdh from #YSVQ#
    • RBS.hps from #H4M9#
    • RBS.phi from #ANNA#
    • RBS.mdh from #KFEY#


15-06-30

RBS.CDS.B0015 in pSB1A3

  • transform ligation products into DH5alpha again

RBS.CDS in pSB1A30

  • control digests on gel
  • results
      • #TUSS#:mdh (expected: 1220 bp / 2037 bp): bands at expected length
      • #TUSS#:hps (expected: 698 bp / 2037 bp): bands at expected length
      • #TUSS#:phi (expected: 617 bp / 2037 bp): bands at expected length
      • #TUSS#:xpk (expected: 2540 bp / 2037 bp): bands at expected length
      • #TUSS#:K30 (expected: 902 bp / 2222 bp): no bands at all
  • ligation of fragments
    • Ligation of #OHOZ#:1 with ...
      • ...#TUSS#:mdh (ML)
      • ...#TUSS#:hps (HL)
      • ...#TUSS#:phi (PL)
      • ...#TUSS#:xpk (XL)

Pipeting volumes for ligation

component volume [µl]
water 10
T4 Ligase Buffer 2
Backbone digest (#OHOZ#:1) 3
part digest (from #TUSS#) 4
T4 Ligase 1
  • ligation products stored in falcon #LIGJ#
  • transformation into BL21 Gold (DE3)

15-07-01

Colony PCR of RBS.CDS.B0015 in pSB1A30

gene / clones tube number / row in gel expected length machine elongation time result
RBS.mdh.B0015 in pSB1A30 clones 6, 8, 10, 11 1-4 1682 2 0'25 no positive clones
RBS.hps.B0015 in pSB1A30 clones 3 5 1160 2 0'25 no positive clones
RBS.phi.B0015 in pSB1A30 clones 2, 4, 6, 7, 8, 13, 17-21 6-16 1079 2 0'25 clones 13, 19, 20 and 21 positive
RBS.xpk.B0015 in pSB1A30 clones 1, 7 17, 18 3002 5 0'45 clone 7 positive

RBS.CDS.B0015 in pSB1A3 (E, F, G, H)

  • make masterplates and overnight cultures

RBS.CDS in pSB1A30 (ML, HL, PL, XL)

  • make masterplates and overnights

15-07-02

RBS.CDS.B0015 in pSB1A30

  • purify plasmid of:
    • xpk clone 7
    • phi clones 13, 19, 20, 21

RBS.CDS.B0015 in pSB1A3 (E, F, G, H)

  • colony PCR
construct ID construct description clone numbers expected length [bp] elongation time result
E RBS.mdh.B0015 in pSB1A3 1-8 1631 0'25 nothing
F RBS.hps.B0015 in pSB1A3 9-16 1109 0'25 nothing
G RBS.phi.B0015 in pSB1A3 17-24 1028 0'25 nothing
H RBS.xpk.B0015 in pSB1A3 25-32 2951 0'45 clone 26 positive
  • purify plasmid and make cryos of positive clones
  • prepare positive clones for sequencing


  • because almost nothing worked: new transformation of ligation products #CAJO#:E/F/G/H
    • 50 µl competent DH5alpha
    • 2 µl ligation product
    • 200 µl SOC media

RBS.CDS in pSB1A30 (ML, HL, PL, XL)

  • colony PCR
construct ID construct description clone numbers expected length [bp] elongation time result
ML RBS.mdh in pSB1A30 33-40 1553 0'25 Clone: 34, 35, 37, 39
HL RBS.hps in pSB1A30 41-48 1031 0'25 Clone: 41 - 48
PL RBS.phi in pSB1A30 49-56 950 0'25 Clone: 49 - 56
XL RBS.xpk in pSB1A30 57-64 2873 0'45 clones 57 and 62 positive
  • purify plasmid and make cryos of positive clones
  • prepare positive clones for sequencing
Construct Clone Plasmid ID Cryo ID Plasmid konzentration Sequencing result
H: RBS.xpk.B0015 in pSB1A3 26 #8ZRY# #84BM# 234.7 VALID
ML: RBS.mdh in pSB1A30 34 #1RZF# #8AZD# 136.5 discard
35 #PZRX# #MB3A# 160.3 VALID
37 #BZBQ# #IGEM# 187.3 VALID
39 #6WTB# #HEXE# 133.8 discard
HL: RBS.hps in pSB1A30 41 #YSAE# #MKCP# 165.4 VALID
42 #T8KB# #DR63# 133.4 discard
43 #H8KF# #F4KN# 158.1 discard
44 #4PAB# #4P9H# 188.0 VALID
PL: RBS.phi in pSB1A30 49 #PEA6# #TFE1# 168.4 VALID
50 #814A# #QHB3# 139.7 discard
51 #AQQ8# #1XAB# 151.2 VALID
52 #VBEZ# #B9XO# 135.2 discard
XL: RBS.xpk in pSB1A30 57 #4AE3# #NF66# 110.5 VALID
62 #1NFT# #ONK4# 268.2 VALID
RBS.xpk.B0015 in pSB1A30 7 #KANH# #CNEK# 110.1 VALID
RBS.phi.B0015 in pSB1A30 13 #RX39# #D8S8# 85.0 VALID
19 #3MNR# #PZ6F# 45.5 discard
20 #XTW8# #E1KA# 58.9 VALID
21 #O3FR# #N9EE# 53.7 discard

15-07-03

  • plasmid purification of all positive clones from yesterday
  • prepare sequencing
  • make LB+A plates
  • masterplates and overnights of unsuccessful RBS.CDS.B0015 in pSB1A3 constructs
  • plan workflow for saturday
    • list of items to take to iAMB:
      • KAPA Mastermix
      • Primers
      • Master plates
      • PCR strips
      • 2-log Ladder
  • edit gel pictures of yesterday (iJR, at home)

15-07-06

as our colony PCRs are very unreliable und the last time, so we will check all the factors that could influence the results (product lengths)

Test with KAPA 2G FAST READY mastermix, each with the same programm:

Step Time Temp
denature 3'00 95
cyc. denature 0'15 95
cyc. anneal 0'15 58
cyc. elongate variable 72
elongate 2'00 72
store forever 16

30 cycles!

  • Test with 1 µl of purified plasmids:
construct ID construct description tube number/row expected length [bp] elongation time cycler result
#CD8B# J04450 in pSB1C3 plasmid 1 1348 0'21 a very strong band with expected length
#KFEY# Xpk in pSB1C3 plasmid 2 2814 0'42 b weak band with expected length + grease
#CT8H# glgAB in pSB1A3 plasmid 3 3986 1'00 c no bands
- Water 4 --- 1'00 c no bands
  • test with 1µl of cryos:
construct ID construct description tube number/row expected length [bp] elongation time cycler result
#W39E# J04450 in pSB1C3 plasmid 5 1348 0'21 a strong band with expected length
#8WY9# Xpk in pSB1C3 plasmid 6 2814 0'42 b no bands
#1Q88# glgAB in pSB1A3 plasmid 7 3986 1'00 c very weak band with expected length
- Water 8 --- 1'00 c weak band at ca. 4000 bp because of pipetting mistake (iCG)

It seems like the mastermix has problems with longer product lengths

  • spread a little bit of the cryos on the corresponding plates, to test colony PCR with colonys

15-07-07

Expression test

  • first expression test of mdh
  • plating of mdh in A30 in BL21 clones:
    • #IGEM#
    • #MB3A#
    • #8ADZ#
    • #HEXE#
  • plating of rfp control:
    • #6DA3#

Colony PCR of template plasmids E, F, G and H

construct ID construct description numbers of clones expected length [bp] elongation time result
E RBS.mdh.B0015 in pSB1A3 27 1631 1'38 clone 6, 7, 8, 10, 17, 25, 27 positive
F RBS.hps.B0015 in pSB1A3 23 1109 1'07 clone 6, 9, 21, 22, 23 positive
G RBS.phi.B0015 in pSB1A3 24 1028 1'07 clone 4, 12, 13, 15, 17, 18, 19 positive
H RBS.xpk.B0015 in pSB1A3 22 2951 2'57 clone 2, 6, 17, 22

15-07-08

  • purify plasmid of constructs E, F, G, H
construct ID construct description clone number cryo ID plasmid ID sequencing results
E RBS.mdh.B0015 in pSB1A3 6 #CHWW# #H39R# VALID
25 #84BB# #RRN3# VALID
27 #WBCK# #L4OY# VALID, but discard to safe space
F RBS.hps.B0015 in pSB1A3 6 #HBDX# #EZWV# VALID
21 #KW16# #8181# VALID
22 #RSWY# #HVXO# VALID, but discard to safe space
G RBS.phi.B0015 in pSB1A3 4 #E89D# #X9WT# discard before sequencing
12 #HWPO# #H9BV# discard before sequencing
18 #F6V1# #DK8F# discard before sequencing
H RBS.xpk.B0015 in pSB1A3 2 #3A1V# #DDT3# discard before sequencing
6 #E63S# #Q9B3# discard before sequencing
22 #S9KK# #3F69# discard before sequencing
  • check sequencing results of sequencing from 15-07-02
  • prepare overnights for expression test of platet cryos:
    • 2x #IGEM# (mdh in pSB1A30)
    • #6DA3# (RFP control)
  • check out results table on top of this page!

15-07-09

Expression Tests

  • we test Mdh (+) induced, Mdh (-) not induced, and Rfp control
time (h)\ Sample (1) Mdh (-) (2) Mdh (+) (0) RFP
0 0.11 0.11 0.12
1 0.16 0.2 0.29
2 0.27 0.47 0.91
2.3 0.37 0.72 1.212
...
  • at 12:20 induced with IPTG, 50 µl (Mdh +) respectively 51 µl (RFP)

15-07-10

  • samples after 5h and after 20 h after induction with IPTG.
  • SDS gel
  • samples were set to OD 10
  • 7 µl of samples were loaded to gel
  • SDS gel showed no significant bands
  • repeat expression test of Mdh with other three genes next week

15-07-13

  • plate cryos on LB+A plates for precultures:
    • mdh: #IGEM#
    • hps: #4P9H#
    • phi: #TFE1#
    • xpk: #ONK4#
    • RFP: #6DA3#

15-07-15

Expression Tests

  • main culture preparation
ID OD Preculture Volume added to 50 ml LB+A [ml]
mdh 1 5.031 1.5
mdh 2 4.872 1.5
hps 1 5.382 1.4
hps 2 5.421 1.4
phi 1 5.826 1.2
phi 2 6.264 1.2
xpk 1 5.442 1.4
xpk 2 5.361 1.4
RFP Control 4.542 1.7
  • main culture OD values
sample OD (1 h) OD (1,5 h) OD (2 h) induced
mdh 1 0.234 0.408 0.568 no
mdh 2 0.254 0.469 0.655 yes
hps 1 0.344 0.624 0.814 no
hps 2 0.305 0.577 0.761 yes
phi 1 0.282 0.526 0.721 yes
phi 2 0.295 0.581 0.776 no
xpk 1 0.270 0.476 0.648 no
xpk 2 0.275 0.494 0.650 yes
Rfp 0.348 0.640 0.816 yes

15-07-16

Expression tests

  • samples taken at 6 h and 21 h after induction with 50 µl IPTG
  • cell pellets were resuspended in 150 µl water
  • suspensions were diluted to OD 10
  • 10 µl SDS added to 50 µl cell suspension (OD 10)
  • incubate at 95 °C for 15 min
  • finished SDS preparations stored in Falcons:
    • mdh: #MDQL#
    • hps: #PBEA#
    • phi: #BHBB#
    • xpk: #ZR8V#

15-07-17

Expression tests

Mdh - SDS gel

  • negative expression test

Hps - SDS gel

  • no positive expression test so far

Phi - SDS gel

  • no positive expression test so far

Xpk - SDS gel

  • positive expression test

15-07-21

Expression tests

Hps - SDS gel

  • no reliable results

Phi - SDS gel

  • no reliable results

New expression tests of Hps and Phi

  • precultures from cryos #4P9H# (hps) and #TFE1# (phi)
  • preculture from cryo #IGEM# (mdh) as control

15-07-22

New expression tests of Hps and Phi

  • make maincultures of Hps and Phi
  • 2 ml samples taken 6 h after induction with IPTG

15-07-23

New expression tests of Hps and Phi

  • prepare samples and load them on the gel
  • run gel at 130 V for 110 min
  • results: gel has bad quality, no clear bands visible

15-07-27

clone mdh, hps and phi into J61002 with constitutive Anderson Promoter J23119 for expression tests with Anderson Promoter 19

restriction

templates:

Cut with XbaI/PstI

  • RBS.mdh in A30 #BZBQ#
  • RBS.hps in A30 #4PAB#
  • RBS.phi in A30 #PEA6#

Products are stored in Falcon #FPTS#

ligation

Backbone J61002.J23119 (cut with SpeI/PstI) in #TF4M# Tube1 (derived from #AHZ6#) is already digested.

Ligate the backbone with each construct, products are stored in #YMVY#


Transformation

Heatshock transformation in BL21; plate on LB+Amp plates.

15-07-28

  • Masterplates of many transformants and overnights of 6 clones each.

15-07-29

Do colony PCR with masterplates with #A9W9# & #XE3D# (chosen annealing temp. 58 °C). (negative with RFP 1143bp, positive Hps 939 bp, positive Phi 858 bp, positive MDH 1461 bp).

  • results of cPCR and red/white screening disagree. In both screenings positive: phi #3, hps #1,2,3,4,5
  • do NEW masterplates and overnights.

15-07-30

Colony PCR on new masterplates

  • this time with one primer that binds on the backbone (#A9W9#) and an insert-specific reverse primer (for mdh: #MSP1#, hps:#HSP2#, phi:#PSP2#)
  • based on the colony PCR, overnights of mdh #4&5, phi #3&5 and hps #1,2,3 were used for cryos and plasmid purification.
  • cryo cultures: #4EMV# #3PVM#; #1MDN# #38L9#; #KL96# #B39O# #BEZO#
  • purified plasmids: #PS1M# #ONMP#; #LSHO# #KQ1W#; #MXYY# #1DPL# #KTZ8#
  • test digest of plasmids with EcoRI. Expected length of hps.J61002: 2731bp; phi.J61002: 2650; mdh.J61002: 3253bp; negative rfp.J61002: 2987 bp

15-08-03

  • run test digest from 30th July on gel: mdh#4, Mdh#5, phi#3, phi#5, hps#1, hps#2, hps#3; all bands at expected lenght
  • repeat colony PCR for hps with #A9W9# and #HSP2#; expected lenght is 621 bp, chosen annealing Temp 58 °C
  • overnight cultures of successful clones

15-08-05

  • Prepare three main cultures (hps #KL96#, mdh #3PVM#, phi #1MDN#). RFP control #13YN# did not grow. We have to use #BYS8#
  • Prepare 2 SDS (12 % for Mdh; 15 % for Hps, Phi) gels for tomorrow.
  • Preculture of #BYS8#

Tomorrow: Prepare cell pellets according to the protocol. Run one gel with Mdh and Rfp as control and a second gel with Hps, Phi and Mdh as control. The SDS-PAGE with Mdh and RFP as control has to be done on Friday.

15-08-06

  • Main culture of the Rfp control (BYS8). OD at 10.45 is 0.3. OD at 11:30 is 0.788. Take 2 2 ml samples at 17:15 and freeze the pellet.
  • Load SDS gel (15 %) with the 6 h-samples of Mdh (#3PVM#), Phi (#1MDN#) and Hps (#KL96#). Gel runs at 120 V for 90 minutes. Expected Mdh 40 kDa; Phi 20 kDa & Hps 22.5 kDa. All samples are negative.
  • Take 22h samples for back-ups and freeze them.
  • Prepare two more SDS gels (12 % for Mdh and Rfp tomorrow and one 15 % as backup for Hps,Phi & Mdh)

15-08-07

Prepare suspensions with OD 0.13 of the samples (6 and 22 hours)

  • Do SDS-PAGE Mdh and Rfp with 12 % acrylamid.
  • Hps, Phi & Mdh with 15 % acrylamid. Results can be found in SDS-Gels 15-08-07.
  • Gel with Hps, Phi & Mdh is completely negative.
  • Gel with Mdh & Rfp has a very strong band in the Rfp 6 h Sample slightly abouve 26 kDa but Rfp has 25 kDa.

Prepare for sequencing: 15-08-07 Sequencing Sheet

sequencing of #PS1M# #ONMP#; #LSHO# #KQ1W#; #MXYY# #1DPL# #KTZ8#

Plated all four genes + RFP in pSB1A30 in BL21 Gold (DE3) cells on LB+A (#IGEM#, #4P9H#, #TFE1#, #ONK4#, #6DA3#)

sequencing results of mdh, phi & hps in J61002

  • #PS1M#: VALID
  • #ONMP#: VALID
  • #LSHO#: VALID
  • #KQ1W#: VALID
  • #MXYY#: VALID
  • #1DPL#: VALID
  • #KTZ8#: VALID

15-08-08

Do overnight cultures from all genes in pSB1A30 (#IGEM#, #4P9H#, #TFE1#, #ONK4#, #6DA3#), cultivation startet 16:30.

15-08-09

Do 50 ml mainculture of A30 constructs (injected with 2 ml overnight culture) in LB media with ampicilin. Incubation started at 10:07. Induce with 60 µl 1000x stock IPTG at 12:15 (OD600 of samples: #IGEM# 0.79; #4P9H# 0.758; #TFE1# 0.871; #ONK4# 0.640; #6DA3# 0.310).

After 6 h (18:15) take 2x2 ml samples of each cultivation and again next morning.

15-08-10

  • Take 2x2 ml (21 h) samples of mainculture.
  • make two 15 % SDS-Gels.

Result:

Xpk, Hps, Phi & Mdh are successfully expressed (Mdh is a bit harder to see)

expression of polycistronic construct

The expression of the polycistronic construct is here.

References

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