View source for Team:Aachen/Notebook/Documentation/Glycogen Single Expression

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{{Team:Aachen/ReadMore|title=Single Expression|link=Team:Aachen/Lab/Glycogen/Synthesis/II._Single_Expression|picture=rmGlycogenSynthesisII|url=/2015/images/0/0c/Aachen_tile_Glycogen_Glycogen_synthesis_single_expression.JPG}}
=Laboratory Notebook=

==15-06-09==
digest glgA/B plasmids ('''stored in #SZKD#''')
{|class="wikitable"
! name !! plasmid ID !! enzyme 1 !! enzyme 2
|-
| A2 (glgA) || #M1M6# || EcoRI || PstI
|-
| B2 (glgB) || #YHT3# || EcoRI || PstI
|-
| pSB1K30   || #PCFX# || EcoRI || PstI
|}

==15-06-10==
* after digest glg A and B:
** A and B will be ligated in pSB1K30 (ligation tubes: A2 and B2)
** '''stored in #QHHR#'''
* transformation into DH5α 

==15-06-11==
* master plates of glgA, glgB in pSB1K30 (LB+K)

==15-06-12==
* colony PCR on glgA/B constructs to identify positive clones
* make overnight cultures of glgA/B constructs

{|class="wikitable"
! clone number !! cryo ID !! plasmid ID !! sequencing result
|-
| glgA in pSB1K30 #2 || #3DPV# || #D6VB# || mRFP
|-
| glgA in pSB1K30 #3 || #4LCM# || #RLDN# || mRFP
|-
| glgB in pSB1K30 #5 || #6YBE# || #ATE6# || pT7 is missing
|}

==15-06-13==
* make cryos of glgA/B constructs (table above)
* plasmid prep of glgA, glgB (table above)

==15-06-15==
* measured concentrations of purified glgA/glgB constructs
* heat shock of glgA (#D6VB#, #RLDN#) and glgB (#ATE6#) in pSB1K30 into BL21(DE3)

==15-06-16==
* BL21 transformation master plates (glgB transformation was not successful)

==15-06-17==
Digestions with 40' digest and 20' inactivation

(previous digests A2, B2, pSB1K30 were discarded from #SZKD# because agarose gels were really ugly)

{|class="wikitable"
! gene !! enzymes !! expectation !! observation !! products
|-
| glgA (#M1M6#) || EcoRI + PstI || ... || confirmed || #SZKD#:A2
|-
| glgB (#YHT3#) || EcoRI + PstI || ... || confirmed || #SZKD#:B2
|-
| pSB1K30 (#PCFX#) || EcoRI + PstI || ... || confirmed but weak || #SZKD#:K30
|}


Ligations and transformations
{|class="wikitable"
! construct !! part I !! part II !! backbone !! product !! host
|-
| glgA in pSB1K30 || #SZKD#:A2 || - || #SZKD#:K30 || #99MC#:LA || BL21
|-
| glgB in pSB1K30 || #SZKD#:B2 || - || #SZKD#:K30 || #99MC#:LB || BL21
|}

==15-06-18==
* make master plates and liquid cultures of transformations (glgA in pSB1K30, glgB in pSB1K30; '''6 liquid, 12 plate''')

==15-06-19==
Colony PCR on glgA,B,C constructs

{|class="wikitable"
! gene / clones !! tube number !! expected length !! machine !! elongation time !! result
|-
| glgA in pSB1K30 clones 1-12 || 49-60 || 1770 || 2 || 1'50" || three okay
|-
| glgB in pSB1K30 clones 1-12 || 61-72 || 2523 || 3 || 3'05" || all negative
|}

Cryos
{|class="wikitable"
! gene / clone !! cryo ID !! plasmid ID !! sequencing result
|-
| glgA in pSB1K30 #2 || #VWSS# || #FHTH# || -
|-
| glgA in pSB1K30 #3 || #W8VB# || #K8DM# || perfect
|-
| glgA in pSB1K30 #6 || #4TPL# || #K3EO# || good, but possibly one (unlikely) insertion
|}

Master plates of glgA, glgB clones that were white under green light.

==15-06-22==
* prep good plasmids of glgA (table above)
* pipette glgA in pSB1K30 for sequencing (#A9W9# and #XE3D#)
* colony PCR on glgA, glgB plates from Saturday (#A9W9# and #XE3D#)

{|class="wikitable"
! gene / clones !! tube number !! expected length !! machine !! elongation time !! result
|-
| glgA in pSB1K30 clones 13-15 || 12-14 || 1821 || 2 || 1'50" || all three are good
|-
| glgB in pSB1K30 clones 13-15 || 15-17 || 2572 || 3 || 3'05" || clone 14 is good
|}

* overnight cultures of good clones from the glgB colony PCR (LB+K)


==15-06-23==
* cryos and plasmid prep from overnight cultures of glgB
* the plasmid prep went wrong and resulted in concentrations <&nbsp;10&nbsp;ng/µl

{|class="wikitable"
! gene / clone !! cryo ID !! plasmid ID !! sequencing result
|-
| glgB in pSB1K30 #14 || #Q6Z1# || #KK8K# || confirmed ligation sites
|}

* overnight cultures of glgB in pSB1K30 from the master plate

==15-06-24==
* in the morning: freeze overnight culture of glgB
* in the afternoon: plasmid prep of glgB (re-used the existing ID from the table of Monday)
* pipette glgB for sequencing

==15-06-26==
Digestions
{|class="wikitable"
! BioBrick in Backbone !! gene !! template ID !! enzymes !! product !! purpose
|-
| K1585312 in pSB1A2 || glgC || #F8Y8# || EcoRI, PstI || #LVVK#:1 || subcloning into pSB1C3, pSB1K30
|-
| pSB1K30 || mRFP || #1K30# || EcoRI, PstI || #LVVK#:4 || target backbone for expression
|-
|}

==15-06-29==
Ligation and Transformation (first try)
{|class="wikitable"
! construct !! gene !! Part I !! Part II !! Backbone !! products !! host !! purpose
|-
| K1585312 in pSB1K30 || glgC || #LVVK#:1 || - || #LVVK#:4 || #RRCV#:2 || BL21 Gold (DE3) || for expression tests
|}
>> #LVVK#:4 (pSB1K30) looked bad on agarose gel! Do the ligation again with pSB1K30 from a different tube!

Ligation (second try, only 1&nbsp;µl of K30)
{|class="wikitable"
! construct !! gene !! Part I !! Part II !! Backbone !! products !! host !! purpose
|-
| K1585312 in pSB1K30 || glgC || #LVVK#:1 || - || #SZKD#:K30 || #RRCV#:2 || BL21 Gold (DE3) || for expression tests
|}
* transformation is planned for the day after

==15-06-30==
* heat shock of K1585312 in pSB1K30 ligation product (5&nbsp;µl #RRCV#:2) into 40&nbsp;µl BL21 Gold (DE3)

==15-07-01==
* master plate of K1585312 in pSB1K30 (BL21 Gold DE3)

==15-07-02==
* colony PCR of K1585312 in pSB1K30 (desired: 1683, negative: 1227)
* <s>overnights of good clones</s>

colony PCR with KAPA 2G

conditions for '''30 cycles''':
{|class="wikitable"
! step !! temperature [°C] !! duration
|-
| initial denaturation || 95 || 3'00"
|-
| denaturation || 95 || 0'15"
|-
| annealing || 58 || 0'15"
|-
| elongation || 72 || 0'25"
|-
| final elongation || 72 || 2'00"
|}


Digestions
{|class="wikitable"
! BioBrick in Backbone !! gene !! template ID !! enzymes !! product !! purpose
|-
| K1585312 in pSB1A2 || glgC || #F8Y8# || EcoRI, PstI || #LVVK#:1 || subcloning into pSB1K30
|-
| pSB1K30 || mRFP || #4K19# || EcoRI, PstI || #LVVK#:4 || target backbone for expression
|-
|}

Ligation
{|class="wikitable"
! construct !! gene !! Part I !! Part II !! Backbone !! products !! host !! purpose
|-
| K1585312 in pSB1K30 || glgC || #LVVK#:1 || - || #LVVK#:4 || #RRCV#:2B || BL21 Gold (DE3) || for expression tests
|}

==15-07-03==
* <s>cryo cultures</s>
* <s>plasmid prep</s>
* <s>pipette for sequencing</s>

==15-07-06==
* <s>transformation of #RRCV#:2B into BL21 Gold (DE3)</s>
* decided to use pSB1A30 because the difference between induced and non-induced pSB1K30 could not be seen on SDS gels

PCR 1 to subclone glgC into pSB1A30: backbone amplification

{|class="wikitable"
! component !! template !! forward primer !! reverse primer !! product length !! purified PCR product ID !! product ID !! tube #
|-
| I13507 in pSB1A30 || #LYZH# || #TSTV# || #FYTO# || 2206 || #T6X9# || #MRVW#:III ||3+4
|}

PfuS PCR

conditions for '''30 cycles''':
{|class="wikitable"
! step !! temperature [°C] !! duration
|-
| initial denaturation || 98 || 2'00"
|-
| denaturation || 98 || 0'30"
|-
| annealing || 64 || 0'30"
|-
| elongation || 72 || 1'10"
|-
| final elongation || 72 || 5'00"
|}

==15-07-08==
* <s>master plate (LB+K+IPTG) of glgC in pSB1K30 transformations</s>

PCR2 to subclone glgC into pSB1A30: glgC amplification

{|class="wikitable"
! component !! template !! forward primer !! reverse primer !! product length !! product ID
|-
| glgC in pSB1A2 || #F8Y8# || #O3M6# || #NBZZ# || 1360 || #MRVW#: I
|}

PfuS PCR

conditions for '''30 cycles''':
{|class="wikitable"
! step !! temperature [°C] !! duration
|-
| initial denaturation || 98 || 3'00"
|-
| denaturation || 98 || 0'30"
|-
| annealing || 58 || 0'30"
|-
| elongation || 72 || 0'45"
|-
| final elongation || 72 || 5'00"
|}

==15-07-09==
PCR product purification

{|class="wikitable"
! PCR product !! purified PCR product
|-
| #MRVW#:I || #XNHT#
|-
| #MRVW#:III || #T6X9#
|}

CPEC assembly of glgC in pSB1A30
{|class="wikitable"
! component !! volume [µl]
|-
| water || 5.1
|-
| Q5 buffer || 5
|-
| DMSO || 0.75
|-
| dNTPs (2 mM) || 5
|-
| Q5 polymerase || 0.5
|-
| pSB1A30 fragment (#T6X9#) || 3.9
|-
| glgC with prefix/suffix #XNHT# || 4.8
|-
| elongation time || 1'47"
|-
| product stored in || #SWMB#:II
|}

5&nbsp;µl #SWMB#:II were heat shocked into BL21 Gold (DE3) and plated on LB+A


==15-07-10==
* make masterplates (LB+A) and overnights of glgC in pSB1A30

==15-07-11==
* cryo cultures 
* freeze culture

{|class="wikitable"
! gene / clone !! cryo ID !! plasmid ID !! sequencing result
|-
| B0034.glgC in pSB1A30 #1 || #HXXD# || - || -
|-
| B0034.glgC in pSB1A30 #2 || #D9TA# || - || -
|-
| B0034.glgC in pSB1A30 #3 || #PX1E# || - || -
|-
| B0034.glgC in pSB1A30 #4 || #KKYF# || - || -
|-
| B0034.glgC in pSB1A30 #5 || #M9EY# || - || -
|-
| B0034.glgC in pSB1A30 #6 || #OHES# || #VMN3# || looks perfect
|-
| B0034.glgC in pSB1A30 #9 || #S6KX# || #YLS8# || no T7 promoter
|-
| B0034.glgC in pSB1A30 #10 || #YNNY# || #8MKM# || no T7 promoter
|-
|}

* all clones except #6 were red

==15-07-13==
* plasmid prep of glgC in pSB1A30 clone #6 in #VMN3#
* new overnights of white clones on masterplate: clone #9 and #10

==15-07-14==
* plasmid prep of glgC in pSB1A30 clone #9+10 (table above)
* all three glgC in pSB1A30 plasmids went into sequencing

==15-07-15==
* analysis of sequencing results
* 2 precultures of glgC in pSB1A30 from clone #6, one of RFP in pSB1A30 #6DA3#

=Expression=
==15-06-30==
'''glgA'''
Plated #W8VB# and #KK16# on LB+K

==15-07-01==
* make overnight precultures from the plate (2x #W8VB#, 1x #KK16# -> A,B,C)
* reserve 3x 500&nbsp;ml shake flasks

==15-07-02==
'''08:50''' inoculate 50&nbsp;ml main cultures LB+K (labelled A,B,C) with 1&nbsp;ml of culture

monitor the OD and induce cultures A+B with IPTG at OD=0.6

'''5 h after induction''' from each culture take 2x 2&nbsp;ml sample (centrifuge, discard supernatant and freeze)

'''10 h after induction''' from each culture take 2x 2&nbsp;ml sample (centrifuge, discard supernatant and freeze)

'''24 h after induction''' from each culture take 2x 2&nbsp;ml sample (centrifuge, discard supernatant and freeze)

{|class="wikitable"
! flask !! combination !! inoculated with (time) !! IPTG induction (time) !! 6 h sample !! 18.5 h sample !! pellet
|-
| A || K1585310 in pSB1K30 induced || 1 ml #W8VB# preculture (08:50) || 50 µl IPTG (15:10) || A6, 20:45 #9E9N# || #TX14# || #F3Y6#
|-
| B || K1585310 in pSB1K30 uninduced || 1 ml #W8VB# preculture (08:50) || not induced || B6, 20:45 #EL3O# || #X4KT# || #KBPA#
|-
| C || I13507 in pSB1K30 induced || 1 ml #KK16# preculture (08:50) || 50 µl IPTG (15:10) || C6, 20:45 #9KA9# || #81RD# || #ZCWT#
|}

OD monitoring (numbers show time in hours)
{|class="wikitable"
! flask/ OD !! 1  !!  2!! 3.167 !! 5.333!! 6.333 !! 7.5 !! 8.5
|-
| A || 0.052 || 0.069 || 0.084 || 0.261 || 1.07 || 1.728 || 1.689
|-
| B || 0.056 || 0.077 || 0.094 || 0.314 || 1.241 || 1.834 || 1.811
|-
| C || 0.024 || 0.027 || 0.036  || 0.116 || 0.406 || 1.178 || 1.225
|}

==15-07-03==
* take the final sample
* prepare SDS-PAGE (cooked samples in #B13F#)
* run SDS-PAGE
 
==15-07-06==
'''glgB''' Plated #Q6Z1# on LB+K

==15-07-07==
* make overnight precultures from the plate (2x Q6Z1, (1x KK16) -> A,B,C)
* reserve 3x 500 ml shake flasks

==15-07-08==
'''08:55''' inoculate 50&nbsp;ml main cultures LB+K (labelled A,B,C) with preculture to an OD of 0.1

monitor the OD and induce cultures A+B with IPTG at OD=0.6

'''5 h after induction''' from each culture take 2x 2&nbsp;ml sample (centrifuge, discard supernatant and freeze)

'''10 h after induction''' from each culture take 2x 2&nbsp;ml sample (centrifuge, discard supernatant and freeze)

'''24 h after induction''' from each culture take 2x 2&nbsp;ml sample (centrifuge, discard supernatant and freeze)

{|class="wikitable"
! flask !! combination !! inoculated with (time) !! IPTG induction (time) !! 5 h sample !! 19.5 h sample !! pellet
|-
| A || K1585311 in pSB1K30 induced || 2.5 ml #Q6Z1# preculture (08:50) || 50 µl IPTG (13:40) || A1, 18:50 #B9CL# || #6W3P# || #WP6P#
|-
| B || K1585311 in pSB1K30 uninduced || 2.5 ml #Q6Z1# preculture (08:50) || not induced || B1, 18:50 #SKMW# || #NM6Q# || #ASLE#
|-
| C || I13507 in pSB1K30 induced || 4.5 ml #KK16# preculture (08:50) || 50 µl IPTG (13:40) || C1, 18:50 #9SZH# || #QEHO# || #1CAP#
|}

OD monitoring (numbers show time in hours)
{|class="wikitable"
! flask/ OD !! 1  !!  2.25 !! 3.2 !! 4.2 !! 5 !! 8.33 !! 9 !! 24.2
|-
| A || 0.147 || 0.190 || 0.285 || 0.481 || 0.666 || 2.95 || 2.15 || 2.5
|-
| B || 0.147 || 0.198 || 0.311 || 0.552 || 0.785 || 2.9 || 2.42 || 2.6
|-
| C || 0.100 || 0.108 || 0.109 || 0.115 || 0.111 || 0.64 || 1.41 || 2.0
|}

==15-07-09==
* cooked SDS samples in #6SOX#
* run SDS page

==15-07-15==
* '''glgC''' overnight of clone # from Masterplate of glgC in pSB1A30

==15-07-16==
'''09:15''' inoculate 25&nbsp;ml main cultures LB+A (labelled A,B,C) with preculture to an OD of 0.1

monitor the OD and induce cultures A+B with IPTG at OD=0.6

'''5 h after induction''' from each culture take 2x 2&nbsp;ml sample (centrifuge, discard supernatant and freeze)

'''10 h after induction''' from each culture take 2x 2&nbsp;ml sample (centrifuge, discard supernatant and freeze)

'''24 h after induction''' from each culture take 2x 2&nbsp;ml sample (centrifuge, discard supernatant and freeze)

{|class="wikitable"
! flask !! combination !! inoculated with (time) !! IPTG induction (time) !! 5 h sample !! 19.5 h sample !! pellet
|-
| A || K1585312 in pSB1A30 induced || 0.65 ml #OHES# (from master plate) preculture A (09:15) || 25 µl IPTG (13:05) || A1, 18:10 #NMHL# || A19, 09:00 #ZXSL# || #AQND#
|-
| B || K1585312 in pSB1A30 uninduced || 0.65 ml #OHES# (from master plate) preculture A (09:15) || not induced || B1, 18:10 #WDQZ# || B19, 09:00 #BSVL# || #XROO#
|-
| C || I13507 in pSB1A30 induced || 0.37 ml #6DA3# preculture (09:15) || 25 µl IPTG (11:30) || C1, 18:10 #H4XD# || C19, 09:00 #EQMY# || #LDVR#
|}

OD monitoring (numbers show time in hours)
{|class="wikitable"
! flask/ OD !! 1 !! 2.25 !! 3 !! 4 !! 6 !! 9 !! 24
|-
| A || 0.132 || 0.199 || 0.293 || 0.469 || 2.8 || 4.21, 4.21, 4.21 || 5.72, 6.03, 5.65
|-
| B || 0.126 || 0.202 || 0.291 || 0.463 || 3.6 || 4.34, 4.32, 4.35 || 5.12, 4.91, 5.05
|-
| C || 0.254 || 1.031 || 1.612 || 4.1 || 7.9 || 5.39, 5.41, 5.42 || 5.38, 5.20, 5.12
|}

==15-07-17==
* cooked SDS samples of glgC expressio are in #FWCT#
* SDS PAGE of glgC expression tests

{{Team:Aachen/Figure|Aachen_15-07-17_glgC_expression_test.png|title=SDS PAGE of glgC expression|subtitle=glgC was expressed in pSB1A30 and compared to mRFP expression. The small arrows point to the GlgC bands of 48&nbsp;kDa. No difference between induced and uninduced was observed. After overnight expression the strong bands were no longer present.|size=medium}}

=References=



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