Student Manual

pGLO Transformation
Lesson 1 Introduction to Transformation
!"#$%&'#()*#+,-#.&((#/012,13#)#/1,405-10#6",."#)'#70"0$&4#$1)"'2,13)$&,"8#90303*01
$%)$#)#70"0#&'#)#/&040#,2#:;<#.%&4%#/1,=&50'#$%0#&"'$1-4$&,"'#2,1#3)6&"7#>4,50'#2,1?#)
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30)"'#4%)"70#4)-'05#*+#70"0'B#)"5#&"=,(=0'#$%0#&"'01$&,"#,2#)#70"0#&"$,#)"#,17)"&'3#&"
,1501#$,#4%)"70#$%0#,17)"&'3C'#$1)&$8#A0"0$&4#$1)"'2,13)$&,"#&'#-'05#&"#3)"+#)10)'#,2
*&,$04%",(,7+8#!"#)71&4-($-10B#70"0'#4,5&"7#2,1#$1)&$'#'-4%#)'#21,'$B#/0'$B#,1#'/,&()70#
10'&'$)"40#4)"#*0#70"0$&4)((+#$1)"'2,1305#&"$,#/()"$'8#!"#*&,10305&)$&,"B#*)4$01&)#4)"#*0#
70"0$&4)((+#$1)"'2,1305#.&$%#70"0'#0")*(&"7#$%03#$,#5&70'$#,&(#'/&(('8#!"#305&4&"0B#5&'0)'0'
4)-'05#*+#50204$&=0#70"0'#)10#*07&""&"7#$,#*0#$10)$05#*+#70"0#$%01)/+D#$%)$#&'B#*+#70"0$&4)((+
$1)"'2,13&"7#)#'&46#/01',"C'#40(('#.&$%#%0)($%+#4,/&0'#,2#$%0#50204$&=0#70"0#$%)$#4)-'0'#$%0
5&'0)'08
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F(-,10'40"$#G1,$0&"#>AFG?8#@%0#10)(H(&20#',-140#,2#$%&'#70"0#&'#$%0#*&,(-3&"0'40"$#I0((+2&'%
!"#$%&"'()*+,%&*'8#A100"#F(-,10'40"$#G1,$0&"#4)-'0'#$%0#I0((+2&'%#$,#2(-,10'40#)"5#7(,.#&"
$%0#5)168#F,((,.&"7#$%0#$1)"'2,13)$&,"#/1,405-10B#$%0#*)4$01&)#0J/10''#$%0&1#"0.(+#)4K-&105
I0((+2&'%#70"0#)"5#/1,5-40#$%0#2(-,10'40"$#/1,$0&"B#.%&4%#4)-'0'#$%03#$,#7(,.#)#*1&((&)"$
7100"# 4,(,1#-"501#-($1)=&,(0$#(&7%$8
!"#$%&'#)4$&=&$+B#+,-#.&((#(0)1"#)*,-$#$%0#/1,40''#,2#3,=&"7#70"0'#21,3#,"0#,17)"&'3#$,
)",$%01#.&$%#$%0#)&5#,2#)#/()'3&58#!"#)55&$&,"#$,#,"0#()170#4%1,3,',30B#*)4$01&)#")$-1)((+
4,"$)&"#,"0#,1#3,10#'3)((#4&14-()1#/&040'#,2#:;<#4)((05#/()'3&5'8#G()'3&5#:;<#-'-)((+
4,"$)&"'#70"0'#2,1#,"0#,1#3,10#$1)&$'#$%)$#3)+#*0#*0"02&4&)(#$,#*)4$01&)(#'-1=&=)(8#!"#")$-10B
*)4$01&)#4)"#$1)"'201#/()'3&5'#*)46#)"5#2,1$%#)((,.&"7#$%03#$,#'%)10#$%0'0#*0"02&4&)(#70"0'8
@%&'#")$-1)(#304%)"&'3#)((,.'#*)4$01&)#$,#)5)/$#$,#"0.#0"=&1,"30"$'8#@%0#1040"$#,44-1H
10"40#,2#*)4$01&)(#10'&'$)"40#$,#)"$&*&,$&4'#&'#5-0#$,#$%0#$1)"'3&''&,"#,2#/()'3&5'8
L&,H9)5C'#-"&K-0#/AMN#/()'3&5#0"4,50'#$%0#70"0#2,1#AFG#)"5#)#70"0#2,1#10'&'$)"40#$,
$%0#)"$&*&,$&4#)3/&4&((&"8#/AMN#)(',#&"4,1/,1)$0'#)#'/04&)(#70"0#107-()$&,"#'+'$03B#.%&4%#4)"
*0#-'05#$,#4,"$1,(#0J/10''&,"#,2#$%0#2(-,10'40"$#/1,$0&"#&"#$1)"'2,1305#40(('8#@%0#70"0#2,1#AFG
4)"#*0#'.&$4%05#,"#&"#$1)"'2,1305 40(('#*+#)55&"7#$%0#'-7)1#)1)*&",'0#$,#$%0#40(('C#"-$1&0"$
305&-38#O0(04$&,"#2,1#40(('#$%)$#%)=0#*00"#$1)"'2,1305#.&$%#/AMN#:;<#&'#)44,3/(&'%05#*+
71,.$%#,"#)"$&*&,$&4#/()$0'8#@1)"'2,1305#40(('#.&((#)//0)1#.%&$0#>.&(5H$+/0#/%0",$+/0?#,"
/()$0'#",$#4,"$)&"&"7#)1)*&",'0B#)"5#2(-,10'40"$#7100"#.%0"#)1)*&",'0#&'#&"4(-505#&"#$%0#
"-$1&0"$#)7)1#305&-38#
E,-#.&((#*0#/1,=&505#.&$%#$%0#$,,('#)"5#)#/1,$,4,(#2,1#/012,13&"7#70"0$&4#$1)"'2,13)H
$&,"8##
E,-1#$)'6#.&((#*0P
Q8##@,#5,#$%0#70"0$&4#$1)"'2,13)$&,"8
R8##@,#50$013&"0#$%0#507100#,2#'-440''#&"#+,-1#022,1$'#$,#70"0$&4)((+#)($01#)"#,17)"&'38#

28

Lesson 1 Focus Questions

!"#$#%&$#%'&()%*+(,-.#$&/-+(,%/"&/%(##.%/+%0#%/"+12"/%/"$+12"%-(%/"#%3$+*#,,%+4%
35&((-(2%&%,*-#(/-4-*%5&0+$&/+$)%-(6#,/-2&/-+(7%8#5+9%&$#%&%4#9%4+$%)+1%/+%3+(.#$%&,%)+1
/&:#%+(%/"#%*"&55#(2#%+4%.+-(2%&%2#(#/-*%/$&(,4+$'&/-+(7%
;-(*#%,*-#(/-4-*%5&0+$&/+$)%-(6#,/-2&/-+(,%&$#%.#,-2(#.%/+%2#/%-(4+$'&/-+(%&0+1/%&
<1#,/-+(=%+1$%4-$,/%,/#3%'-2"/%0#%/+%4+$'15&/#%&%<1#,/-+(%4+$%/"-,%-(6#,/-2&/-+(7
Consideration 1: Can I Genetically Transform an Organism? Which
Organism?
>7%%!+%2#(#/-*&55)%/$&(,4+$'%&(%#(/-$#%+$2&(-,'=%)+1%'1,/%-(,#$/%/"#%(#9%2#(#%-(/+%#6#$)
*#55%-(%/"#%+$2&(-,'7%?"-*"%+$2&(-,'%-,%0#//#$%,1-/#.%4+$%/+/&5%2#(#/-*%/$&(,4+$'&/-+(@
+(#%*+'3+,#.%+4%'&()%*#55,=%+$%+(#%*+'3+,#.%+4%&%,-(25#%*#55A

B7%%;*-#(/-,/,%+4/#(%9&(/%/+%:(+9%-4%/"#%2#(#/-*&55)%/$&(,4+$'#.%+$2&(-,'%*&(%3&,,%-/,%(#9
/$&-/,%+(%/+%-/,%+44,3$-(2%&(.%41/1$#%2#(#$&/-+(,7%!+%2#/%/"-,%-(4+$'&/-+(=%9"-*"%9+15.%0#
&%0#//#$%*&(.-.&/#%4+$%)+1$%-(6#,/-2&/-+(=%&(%+$2&(-,'%-(%9"-*"%#&*"%(#9%2#(#$&/-+(%
.#6#5+3,%&(.%$#3$+.1*#,%<1-*:5)=%+$%+(#%9"-*"%.+#,%/"-,%'+$#%,5+95)A

C7%%;&4#/)%-,%&(+/"#$%-'3+$/&(/%*+(,-.#$&/-+(%-(%*"++,-(2%&(%#D3#$-'#(/&5%+$2&(-,'7%?"&/
/$&-/,%+$%*"&$&*/#$-,/-*,%,"+15.%/"#%+$2&(-,'%"&6#%E+$%(+/%"&6#F%/+%0#%,1$#%-/%9-55%(+/%"&$'
)+1%+$%/"#%#(6-$+('#(/A

G7%%8&,#.%+(%/"#%&0+6#%*+(,-.#$&/-+(,=%9"-*"%9+15.%0#%/"#%0#,/%*"+-*#%4+$%&%2#(#/-*%
/$&(,4+$'&/-+(H%&%0&*/#$-1'=%#&$/"9+$'=%4-,"=%+$%'+1,#A%I#,*$-0#%)+1$%$#&,+(-(27%

29

Consideration 2: How Can I Tell if Cells Have Been Genetically

Transformed?
!"#$%%&'($'&'("&)*$%&*+&)","'-#&'.$,/+*.0$'-*,&-/&'*&#($,)"&$,&*.)$,-/01/&'.$-'/
23(",*'43"56&7"+*."&$,4&#($,)"&-,&'("&3(",*'43"&*+&$,&*.)$,-/0&#$,&8"&9"'"#'"9:&$
'(*.*;)(&"<$0-,$'-*,&*+&-'/&,$';.$%&23."='.$,/+*.0$'-*,5&3(",*'43"&0;/'&8"&0$9"6
>**?&$'&'("&#*%*,-"/&*+&!"#$%&'#*,&4*;.&/'$.'".&3%$'"/6&>-/'&$%%&*8/".@$8%"&'.$-'/&*.&
#($.$#'".-/'-#/&'($'&#$,&8"&9"/#.-8"9A
B("&+*%%*C-,)&3."='.$,/+*.0$'-*,&*8/".@$'-*,/&*+&!"#$%&' 0-)('&3.*@-9"&8$/"%-,"&9$'$&'*
0$?"&."+".",#"&'*&C(",&$''"03'-,)&'*&9"'".0-,"&-+&$,4&)","'-#&'.$,/+*.0$'-*,&($/
*##;.."96
$5 D;08".&*+&#*%*,-"/
85 E-F"&*+&A

G5&'("&%$.)"/'&#*%*,4
H5&'("&/0$%%"/'&#*%*,4
I5&'("&0$J*.-'4&*+&#*%*,-"/

#5 K*%*.&*+&'("&#*%*,-"/
95 L-/'.-8;'-*,&*+&'("&#*%*,-"/&*,&'("&3%$'"
"5 M-/-8%"&$33"$.$,#"&C(",&@-"C"9&C-'(&;%'.$@-*%"'&2NM5&%-)('
+5 B("&$8-%-'4&*+&'("&#"%%/&'*&%-@"&$,9&."3.*9;#"&-,&'("&3."/",#"&*+&$,&$,'-8-*'-#&/;#(&$/
$03-#-%%-,
G. L"/#.-8"&(*C&4*;&#*;%9&;/"&'C*&>7O$)$.&3%$'"/:&/*0"&!"#$%&' $,9&/*0"&$03-#-%%-,&'*
9"'".0-,"&(*C&!"#$%&' #"%%/&$."&$++"#'"9&84&$03-#-%%-,6&&

H6 P($'&C*;%9&4*;&"<3"#'&4*;.&"<3".-0",'$%&."/;%'/&'*&-,9-#$'"&$8*;'&'("&"++"#'&*+
$03-#-%%-,&*,&'("&!"#$%&' #"%%/Q

30

Consideration 3: The Genes

!"#"$%&'$()#*+,(-)$%,#'%#.,/."*'$0"'%#*"($%,#',+'*,-"'#"1'234'%#$,'$0"'!"#$%&'
&"//*5'6#')77%$%,#'$,',#"'/)(8"'&0(,-,*,-"9':)&$"(%)',+$"#'&,#$)%#',#"',('-,("'*-)//
&%(&;/)('<%"&"*',+'234'&)//"7'</)*-%7*5'=/)*-%7'234';*;)//>'&,#$)%#*'8"#"*'+,('-,("
$0)#',#"'$()%$5'?&%"#$%*$*'&)#';*"')'<(,&"**'&)//"7'8"#"$%&'"#8%#""(%#8'$,'%#*"($'8"#"*
&,7%#8'+,('#"1'$()%$*'%#$,')'</)*-%75'6#'$0%*'&)*"9'$0"'<!@A'</)*-%7'&)((%"*'$0"'!B=
8"#"'$0)$'&,7"*'+,('$0"'8(""#'+/;,("*&"#$'<(,$"%#')#7')'8"#"'C(&)D'$0)$'&,7"*'+,(')'
<(,$"%#'$0)$'8%."*'$0"':)&$"(%)'("*%*$)#&"'$,')#')#$%:%,$%&5'E0"'8"#"$%&)//>'"#8%#""("7
</)*-%7'&)#'$0"#':"';*"7'$,'8"#"$%&)//>'$()#*+,(-':)&$"(%)'$,'8%."'$0"-'$0%*'#"1'$()%$5

pGLO plasmid DNA

GFP
Cell
wall

Flagellum

Bacterial
chromosomal
DNA

Pore

Beta-lactamase
(antibiotic resistance)

Consideration 4: The Act of Transformation

E0%*'$()#*+,(-)$%,#'<(,&"7;("'%#.,/."*'$0(""'-)%#'*$"<*5'E0"*"'*$"<*')("'%#$"#7"7'$,
%#$(,7;&"'$0"'</)*-%7'234'%#$,'$0"'!"#$%&' &"//*')#7'<(,.%7"')#'"#.%(,#-"#$'+,(''$0"'&"//*
$,'"F<("**'$0"%('#"1/>')&G;%("7'8"#"*5
!"#$"%&#'(&#)*+,#)-./$01#234#'(5"67(#'(&#8&--#$&$95.:&#;"6#<0--=
H5 I*"')'$()#*+,(-)$%,#'*,/;$%,#',+'J)J/K C&)/&%;-'&0/,(%7"D
K5 J)((>',;$')'<(,&"7;("'("+"(("7'$,')*'0")$'*0,&L
>"5#'5.:/?"5$&1#8&--/#'"#75"<#0:#'(&#)5&/&:8&#"?#.$)080--0:#;"6#$6/'=
M5 =(,.%7"'$0"-'1%$0'#;$(%"#$*')#7')'*0,($'%#&;:)$%,#'<"(%,7'$,':"8%#'"F<("**%#8'$0"%(
#"1/>')&G;%("7'8"#"*

31

Lesson 2 Transformation Laboratory

!) List
Workstation Check (!
!"#$%&"$'()*)+",- !"#$%&"'()"*+)(,--'&$()#."#)(./,'+)0$)-%$($*#)"#)1/,%)2/%3(#"#&/*)-%&/%
#/)0$4&**&*4)#.&()'"0)"%$)'&(#$+)0$'/25
.)#/0,)%&"$'()*)+",(
!"#$%&'#(#"%#$%)-'"#$
7/,%$+)"4"%)-'"#$()86)9:;)<)9:=">-;)6)9:=">-="%"?
A%"*(B/%>"#&/*)(/',#&/*
9:)*,#%&$*#)0%/#.
C*/D,'"#&/*)'//-()
7&-$#(
H/">)>&D%/#,0$)./'+$%=B'/"#
I/*#"&*$%)B,'')/B)D%,(.$+)&D$)8B/">)D,-?
!"%3&*4)-$*
I/-1)/B)J,&D3)K,&+$

1#230$%$04#+$0/
6
@
6
6
E)86)-3)/B)6F?
G
6
6
6
6

!6
5!
"
"
"
"
"
"
"
"
"
"

7,()$#8)"$9(%58"22",6%&"$'()*)+",5)L)'&(#)/B)>"#$%&"'(;)(,--'&$()"*+)$M,&->$*#)#."#
(./,'+)0$)-%$($*#)"#)")D/>>/*)'/D"#&/*)#/)0$)"DD$(($+)01)1/,%)#$">)&()"'(/)'&(#$+)0$'/25
N$.1+%"#$+)-K9O)-'"(>&+
@<QI)2"#$%)0"#.)"*+)#.$%>/>$#$%
REQI)&*D,0"#/%
8/-#&/*"';)($$)K$*$%"')9"0/%"#/%1 S3&''(TC*D,0"#&/*?

32

6)P&"'
6

"
"

"

Transformation Procedure

-pGLO

+pGLO

!" #$%&'()*&(+'),&-(./+0)(1&,1(12%&(!"#$% $*-($*)13&0(&"#$%"(#$%&'(%)13(12%&,(4/13

5)20(60)278,(*$.&"(9'$+&(13&.(/*(13&(:)$.(12%&(0$+;"

<" =7&*(13&(12%&,($*->(2,/*6($(,1&0/'&(10$*,:&0(7/7&1>(10$*,:&0(<?@(A'():(10$*,:)0.$1/)*(
,)'21/)*(BC$C'<D(/*1)(&$+3(12%&"

Transformation Solution

33

-pGLO

+pGLO

250 l

!" #$%&'()*'()+,'-(./(0&'"(

Ice

+pGLO

1" 2-'(%(-)'30$'($..4().(40&5(+4(!"#$%&'(")*'*%+"*,"-!).(/$!"63.7(8.+3(-)%3)'3(4$%)'"(#0&5(+4
)*'(01234 )+,'(%/9(077'3-'()*'($..4(0/).()*'()3%/-6.37%)0./(-.$+)0./(%)()*'(,.)).7(.6
)*'()+,'"(:40/()*'($..4(,');''/(8.+3(0/9'<(60/='3(%/9()*+7,(+/)0$()*'('/)03'(&.$./8(090-4'3-'9(0/()*'()3%/-6.37%)0./(-.$+)0./(>;0)*(/.(6$.%)0/=(&*+/5-?"(#$%&'()*'()+,'(,%&5(0/
)*'()+,'(3%&5(0/()*'(0&'"(2-0/=(%(/';(-)'30$'($..4@(3'4'%)(6.3()*'(51234 )+,'"((

(+pGLO)

(-pGLO)

+pGLO

A" B<%70/'()*'(4CDE(FGH(-.$+)0./(;0)*()*'(2I($%74"(G.)'(8.+3(.,-'3J%)0./-"
K77'3-'(%(%(6"#.(/$'("'**1 0/).()*'(4CDE(4$%-709(FGH(-).&5()+,'"(L0)*93%;(%
$..46+$"(M*'3'(-*.+$9(,'(%(60$7(.6(4$%-709(-.$+)0./(%&3.--()*'(30/="(M*0-(0-(-070$%3().
-''0/=(%(-.%48(60$7(%&3.--(%(30/=(6.3(,$.;0/=(-.%4(,+,,$'-"(N0<()*'($..46+$(0/).()*'
&'$$(-+-4'/-0./(.6()*'(01234 )+,'"(O$.-'()*'()+,'(%/9(3')+3/(0)().()*'(3%&5(./(0&'"
H$-.(&$.-'()*'(51234 )+,'"(7*"%*. %99(4$%-709(FGH().()*'(51234 )+,'"(L*8(/.)P(

pGLO Plasmid DNA

(+pGLO)

34

(-pGLO)

!" #$%&'()*+),*+)&'*-+.$+/%*+0.1+23+4/$&)*-"+5(6*+-&1*+).+7&-,+),*+)&'*-+(88+),*+9(:+;.9$
/$+),*+1(%6+-.+),*+'.)).4+.0+),*+)&'*-+-)/%6+.&)+($;+4(6*+%.$)(%)+9/),+),*+/%*"

Rack

Ice

<" =,/8*+),*+)&'*-+(1*+-/))/$>+.$+/%*?+8('*8+:.&1+0.&1+@A+$&)1/*$)+(>(1+78()*-+.$+),*+'.)).4
B$.)+),*+8/;C+(-+0.88.9-D
E

@('*8+.$*+!"#$%& 78()*D

'(&)!*

@('*8+),*+!"#$%&#$+$ 78()*D

'(&)!*

@('*8+),*+.),*1+!"#$%& 78()*D ,(&)!*

@('*8+),*+!" 78()*D

pGLO

,(&)!*

pGLO

LB/
amp/ara

LB/amp

pGLO

pGLO

LB/amp

LB

F" -.$/(012345 G-/$>+),*+0.(4+1(%6+(-+(+,.8;*1?+)1($-0*1+'.),+),*+BHC+7I@J+($;+BKC

7I@J+)&'*-+/$).+),*+9()*1+'(),?+-*)+()+LM.N?+0.1+*O(%)8:+P3+-*%.$;-"+5(6*+-&1*+).
7&-,+),*+)&'*-+(88+),*+9(:+;.9$+/$+),*+1(%6+-.+),*+'.)).4+.0+),*+)&'*-+-)/%6+.&)+($;
4(6*+%.$)(%)+9/),+),*+9(14+9()*1"
=,*$+),*+P3+-*%.$;-+(1*+;.$*?+78(%*+'.),+)&'*-+'(%6+.$+/%*"+Q.1+),*+'*-)+)1($-0.14()/.$
1*-&8)-?+),*+)1($-0*1+01.4+),*+/%*+B3RNC+).+LMRN+($;+),*$+'(%6+).+),*+/%*+4&-)+'*+1(7/;"
#$%&'()*+)&'*-+.$+/%*+0.1+M+4/$&)*-"

Water bath
Ice

42C for 50 seconds

35

Ice

!" #$%&'$()*$(+,-.(-&/),0/0/1()*$()23$4(5+&%()*$(0-$(,/6(78,-$(&/()*$(3$/-*()&7"(97$/
,()23$(,/6:(240/1(,(/$;(4)$+08$(707$):(,66(<=>(?8(&5(@A(/2)+0$/)(3+&)*()&()*$()23$(,/6
+$-8&4$(0)"(#$7$,)(;0)*(,(/$;(4)$+08$(707$)(5&+()*$(&)*$+()23$"(B/-23,)$()*$()23$4(5&+
C>(%0/2)$4(,)(+&&%()$%7$+,)2+$"(

-pGLO

+pGLO

250 l

LB broth

-pGLO

+pGLO

C>" D,7()*$(-8&4$6()23$4(;0)*(E&2+(50/1$+()&(%0F"(G40/1(,(/$;(4)$+08$(707$)(5&+($,-*()23$:
707$)(C>>(?8(&5()*$()+,/45&+%,)0&/(,/6(-&/)+&8(4247$/40&/4(&/)&()*$(,77+&7+0,)$(/2)+0$/)
,1,+(78,)$4"(

100 l

amp

+p

GL

LB
/amp/ara

-p

GL

LB/

GL

LB/

-p

amp

36

GL
LB

Control plates

Transformation plates

+p

!!" !"#$%$&#'$"(#)*+#$+,,-$.,)$#%/0$-+%(#"#$%&'()#*+'#,-,%'.,/0.,#'1'.23#(&0-.)#*+'#,-&4(5'
04#*+'#67#.-*&/'.*#(8(&#93#:-/5;23#,;(*/.8#*+'#42(*#,-&4(5'#04#(#.'<#,*'&/2'#200%#9(5;#(.)
40&*+#(5&0,,#*+'#%2(*'#,-&4(5'"#=>#?>@#ABC$$#@>>#=CCA#D?@>#@EC#FGFB"

+p

amp

GL

LB
/amp/ara

-p

GL

LB/

GL

LB/

amp

-p

+p

GL
LB

!H" $*(5;#-%#30-&#%2(*',#(.)#*(%'#*+'I#*08'*+'&"#A-*#30-&#8&0-%#.(I'#(.)#52(,,#%'&/0)
0.#*+'#90**0I#04#*+'#,*(5;#(.)#%2(5'#*+'#,*(5;#04#%2(*',#1-"*2#$2,'& /.#*+'#JKLM
/.5-9(*0&#-.*/2#*+'#.'N*#)(3"#

37

Lesson 2 Review Questions

Name ___________________

!"#$%"&'$(("')*+,&-.).&.+-&.+.(/0*+,&/$1%&%"21()2&.+23"%&)4"&#$(($3*+,&51"2)*$+26
76 8+&34*'4&$#&)4"&9(.)"2&3$1(-&/$1&":9"')&)$&#*+-&;.')"%*.&<$2)&(*="&)4"&$%*,*+.(&
+$+>)%.+2#$%<"-&!"#$%&' '$($+*"2&/$1&*+*)*.((/&$;2"%?"-@&A:9(.*+&/$1%&9%"-*')*$+26

B6 C#&)4"%"&.%"&.+/&,"+")*'.((/&)%.+2#$%<"-&;.')"%*.(&'"((2D&$+&34*'4&9(.)"E2F&3$1(-&)4"/
<$2)&(*="(/&;"&($'.)"-@&A:9(.*+&/$1%&9%"-*')*$+26

G6 H4*'4&9(.)"2&24$1(-&;"&'$<9.%"-&)$&-")"%<*+"&*#&.+/&,"+")*'&)%.+2#$%<.)*$+&4.2
$''1%%"-@&H4/@

I6 H4.)&*2&<".+)&;/&.&'$+)%$(&9(.)"@&H4.)&91%9$2"&-$"2&.&'$+)%$(&2"%?"@

38

Lesson 3 Data Collection and Analysis

A. Data Collection
!"#$%&$'()$'%$#*+(#',-*'-"(./0$1'2%-3'()$'(%.0#2-%3.(/-0'+."'*01$%'0-%3.+'%--3'+/4)(/045
6)$0'(*%0'-*('()$'+/4)(#'.01')-+1'()$'*+(%.&/-+$('+/4)('-&$%'()$'7+.($#5'
85 9.%$2*++,'-"#$%&$'.01'1%.:':).(',-*'#$$'-0'$.;)'-2'()$'2-*%'7+.($#5'<*(',-*%'1%.:/04#
/0'()$'1.(.'(."+$'/0'()$';-+*30'-0'()$'%/4)(5'=$;-%1',-*%'1.(.'(-'.++-:',-*'(-';-37.%$
-"#$%&.(/-0#'-2'()$'>!"#$%&?';$++#':/()',-*%'-"#$%&.(/-0#'2-%'()$'0-0@(%.0#2-%3$1'
!"#$%&'5'A%/($'1-:0'()$'2-++-:/04'-"#$%&.(/-0#'2-%'$.;)'7+.($5
B5''C-:'3*;)'".;($%/.+'4%-:()'1-',-*'#$$'-0'$.;)'7+.($D'%$+.(/&$+,'#7$.E/04F''
G5''A).(';-+-%'.%$'()$'".;($%/.F''
H5''C-:'3.0,'".;($%/.+';-+-0/$#'.%$'-0'$.;)'7+.($'I;-*0('()$'#7-(#',-*'#$$J5'

&'()*+,-./0(

3*,0(4/*5,-./0"#2,-)(

K7LM!
MNO.37

K7LM!
MNO.37O.%.

&'()*+,-./0(

1/0-*/2"#2,-)(

@7LM!
MNO.37

@7LM!
MN

39

B. Analysis of Results
!"#$%&'($&)$*'+'$','(-./.$)&0$+"/.$/,1#.+/%'+/&,$/.$+&$*#+#02/,#$/)$%#,#+/3$+0',.)&02'+/&,
"'.$&33400#*5
65 7"/3"$&)$+"#$+0'/+.$+"'+$-&4$&0/%/,'((-$&8.#01#*$)&0$!"#$%&' */*$,&+$.##2$+&$8#3&2#
'(+#0#*9$$:,$+"#$.;'3#$8#(&<$(/.+$+"#.#$4,+0',.)&02#*$+0'/+.$',*$"&<$-&4$'00/1#*$'+$+"/.
','(-./.$)&0$#'3"$+0'/+$(/.+#*5
!"#$#%&'()"&#)

*%&'+,#,(-.(-/,0"1&)#-%,

=5 >)$+"#$!"#$%&' +0'/+.$-&4$&0/%/,'((-$,&+#*?$<"/3"$.##2$,&<$+&$8#$./%,/)/3',+(-$*/))#0#,+
')+#0$;#0)&02/,%$+"#$+0',.)&02'+/&,$;0&3#*40#9$@/.+$+"&.#$+0'/+.$8#(&<$',*$*#.30/8#$+"#
3"',%#.$+"'+$-&4$&8.#01#*5
203()"&#)

!/,0"104(56&%$0

A5 :)$+"#$%#,#+/3'((-$+0',.)&02#*$3#((.$"'1#$'3B4/0#*$+"#$'8/(/+-$+&$(/1#$/,$+"#$;0#.#,3#$&)$+"#
',+/8/&+/3$'2;/3/((/,?$+"#,$<"'+$2/%"+$8#$/,)#00#*$'8&4+$+"#$&+"#0$%#,#.$&,$+"#$;('.2/*
+"'+$-&4$4.#*$/,$-&40$+0',.)&02'+/&,$;0&3#*40#9$

C5 D0&2$+"#$0#.4(+.$+"'+$-&4$&8+'/,#*?$"&<$3&4(*$-&4$;0&1#$+"'+$+"#$3"',%#.$+"'+$&33400#*
<#0#$*4#$+&$+"#$;0&3#*40#$+"'+$-&4$;#0)&02#*9

40

Lesson 3 Review Questions

Name _____________________

Whats Glowing?
!"#$#"%&'()*+),-#.()),#+'%'(#/*#'0*)(1)2#/,#-3)#!"#$%&' +'%',/)*#-3),#$#,)4#5&)*-/',
6/.3-#4)%%#0)#($/*)27#893$-#$()#-3)#-4'#:'**/0%)#*'&(+)*#'"#"%&'()*+),+)#4/-3/,#-3)
+'%',/)*#43),#);:'*)2#-'#<=#%/.3->?
@;:%$/,A
BC D)+$%%#43$-#E'&#'0*)(1)2#43),#E'&#*3/,)2#-3)#<=#%/.3-#',-'#$#*$6:%)#'"#'(/./,$%
:FGH#:%$*6/2#IJK#$,2#2)*+(/0)#E'&(#'0*)(1$-/',*C

LC 93/+3#'"#-3)#-4'#:'**/0%)#*'&(+)*#'"#-3)#"%&'()*+),+)#+$,#,'4#0)#)%/6/,$-)2>

MC 93$-#2')*#-3/*#'0*)(1$-/',#/,2/+$-)#$0'&-#-3)#*'&(+)#'"#-3)#"%&'()*+),+)>##

NC I)*+(/0)#-3)#)1/2),+)#-3$-#/,2/+$-)*#43)-3)(#E'&(#$--)6:-#$-#:)("'(6/,.#$#.),)-/+
-($,*"'(6$-/',#4$*#*&++)**"&%#'(#,'-#*&++)**"&%C

41

Lesson 3 Review Questions

Name ____________________

The Interaction between Genes and Environment

!""#$%&%'($%)$*"+,$-"+,$./%)012$3"$*"+$"410,50$1"60$!"#$%&' &,"7'(&$"($)80$!9$./%)0$)8%)
:"01$(")$;"()%'($%6.';'//'($",$%,%4'("10<

=2 >,"6$*"+,$,01+/)1?$;%($*"+$)0//$'-$)8010$4%;)0,'%$%,0$%6.';'//'($,01'1)%()$4*$/""#'(&$%)
)806$"($)80$!9$./%)0<$$@A./%'($*"+,$%(170,2

B2 C"7$7"+/:$*"+$;8%(&0$)80$4%;)0,'%D1$0(5',"(60()E)80$./%)0$)80*$%,0$&,"7'(&$"(E)"
401)$)0//$'-$)80*$%,0$%6.';'//'($,01'1)%()<

F2 G0,*$"-)0($%($",&%('16D1$),%')1$%,0$;%+10:$4*$%$;"64'(%)'"($"-$')1$&0(01$%(:$')1$0(5',"(60()2
H8'(#$%4"+)$)80$&,00($;"/",$*"+$1%7$'($)80$&0(0)';%//*$),%(1-",60:$4%;)0,'%I
%2

J8%)$)7"$-%;)",1$6+1)$40$.,010()$'($)80$4%;)0,'%D1$0(5',"(60()$-",$*"+$)"$100$)80
&,00($;"/",<$KC'()I$$"(0$-%;)",$'1$'($)80$./%)0$%(:$)80$")80,$-%;)",$'1$'($8"7$*"+
/""#$%)$)80$4%;)0,'%L2

42

J8%)$:"$*"+$)8'(#$0%;8$"-$)80$)7"$0(5',"(60()%/$-%;)",1$*"+$/'1)0:$%4"50$%,0
:"'(&$)"$;%+10$)80$&0(0)';%//*$),%(1-",60:$4%;)0,'%$)"$)+,($&,00(<

;2

J8%)$%:5%()%&0$7"+/:$)80,0$40$-",$%($",&%('16$)"$40$%4/0$)"$)+,($"($",$"--$.%,)';+/%,
&0(01$'($,01."(10$)"$;0,)%'($;"(:')'"(1<

42

Lesson 4 Extension Activity: Calculate Transformation

Efficiency
!"#$%&'()%)*+,%-&%).-+%-&/'+)-0*)-"&%1-22%3'%)"%2'*$&%."1%)"%4')'$5-&'%).'%'()'&)%)"
1.-6.%7"#%0'&')-6*227%)$*&+8"$5'4%!"#$%&' 6'22+9%:.-+%;#*&)-)*)-/'%5'*+#$'5'&)%-+%$'8'$$'4
)"%*+%).'%)$*&+8"$5*)-"&%'88-6-'&679%%
<&%5*&7%'(='$-5'&)+>%-)%-+%-5="$)*&)%)"%0'&')-6*227%)$*&+8"$5%*+%5*&7%6'22+%*+%="++-32'9%?"$
'(*5=2'>%-&%+"5'%)7='+%"8%0'&'%).'$*=7>%6'22+%*$'%6"22'6)'4%8$"5%).'%=*)-'&)>%)$*&+8"$5'4%-&%).'
2*3"$*)"$7>%*&4%).'&%=#)%3*6,%-&)"%).'%=*)-'&)9%:.'%5"$'%6'22+%).*)%*$'%)$*&+8"$5'4%)"%=$"4#6'
).'%&''4'4%=$")'-&>%).'%5"$'%2-,'27%).*)%).'%).'$*=7%1-22%1"$,9%:.'%)$*&+8"$5*)-"&%'88-6-'&67%-+
6*26#2*)'4%)"%.'2=%+6-'&)-+)+%4')'$5-&'%."1%1'22%).'%)$*&+8"$5*)-"&%-+%1"$,-&09
!"#$!%&'
!"#%*$'%*3"#)%)"%6*26#2*)'%).'%)$*&+8"$5*)-"&%'88-6-'&67>%1.-6.%0-/'+%7"#%*&%-&4-6*)-"&
"8%."1%'88'6)-/'%7"#%1'$'%-&%0'))-&0%@AB%5"2'6#2'+%-&)"%3*6)'$-*2%6'22+9%:$*&+8"$5*)-"&
'88-6-'&67%-+%*%&#53'$9%<)%$'=$'+'&)+%).'%)")*2%&#53'$%"8%3*6)'$-*2%6'22+%).*)%'(=$'++%).'
0$''&%=$")'-&>%4-/-4'4%37%).'%*5"#&)%"8%@AB%#+'4%-&%).'%'(='$-5'&)9%C<)%)'22+%#+%).'%)")*2
&#53'$%"8%3*6)'$-*2%6'22+%)$*&+8"$5'4%37%"&'%5-6$"0$*5%"8%@AB9D%:.'%)$*&+8"$5*)-"&%
'88-6-'&67%-+%6*26#2*)'4%#+-&0%).'%8"22"1-&0%8"$5#2*E
:$*&+8"$5*)-"&%'88-6-'&67%F%%:")*2%&#53'$%"8%6'22+%0$"1-&0%"&%).'%*0*$%=2*)'
B5"#&)%"8%@AB%+=$'*4%"&%).'%*0*$%=2*)'%C-&%G0D
:.'$'8"$'>%3'8"$'%7"#%6*&%6*26#2*)'%).'%'88-6-'&67%"8%7"#$%)$*&+8"$5*)-"&>%7"#%1-22
&''4%)1"%=-'6'+%"8%-&8"$5*)-"&E
()* !"#$+,+%-$./01#2$,3$42##.$3-/,2#&5#.+$5,-,.6#&$42,76.4$,.$8,/2$9:;%0<;%2%
<-%+#=
(>* !"#$+,+%-$%0,/.+$,3$<?9@$<-%&06A$BCD$6.$+"#$1%5+#26%-$5#--&$&<2#%A$,.$+"#
9:;%0<;%2%$<-%+#=

43

1. Determining the Total Number of Green Fluorescent Cells

!"#$%&'()*&+,-#./-#*#&/"#0%&1%#*&#&23&"45607&8#$6&$("(1'&(1&06%&/"#0%&$#1&9%&#::).%;&0(
9%&;%*4<%;&=*(.&#&:415"%&$%""7&>:&41;4<4;)#"&$%"":&*%/*(;)$%?&.(*%&#1;&.(*%&$%"":&#*%
=(*.%;&#1;&;%<%"(/&410(&@6#0&4:&0%*.%;&#&$("(1'7&A6%&.(:0&;4*%$0&@#'&0(&;%0%*.41%&06%
!"!#$%&'()*+ ",%-+**&%,$'"+*./*&!%/*$$. 4:&0(&$()10&06%&$("(14%:&(1&06%&/"#0%7
810%*&06#0&1).9%*&6%*%&

A(0#"&1).9%*&(=&$%"":&L
TTTTTTT

2. Determining the Amount of pGLO DNA in the Bacterial Cells Spread on

the LB/amp/ara Plate
B%&1%%;&0@(&/4%$%:&(=&41=(*.#04(1&0(&=41;&()0&06%&#.()10&(=&/C+D&EF>&41&06%&9#$0%*4#"
$%"":&:/*%#;&(1&06%&+,-#./-#*#&/"#0%&41&064:&%G/%*4.%107&H#I&B6#0&@#:&06%&0(0#"&#.()10&(=
EF>&@%&9%5#1&06%&%G/%*4.%10&@406?&#1;&H9I&B6#0&=*#$04(1&(=&06%&EF>&H41&06%&9#$0%*4#I&
#$0)#""'&5(0&:/*%#;&(10(&06%&+,-#./-#*#&/"#0%:7&
D1$%&'()&$#"$)"#0%&064:&;#0#?&'()&@4""&1%%;&0(&.)"04/"'&06%&!"!#$%#("'&!%",%0123%456
):%;&41&064:&%G/%*4.%10&9'&06%&,+#/!7"&%",%456 '()&:/*%#;&(1&06%&+,-#./-#*#&/"#0%7&A6%
#1:@%*&0(&064:&.)"04/"4$#04(1&@4""&0%""&'()&06%&#.()10&(=&/C+D&EF>&41&06%&9#$0%*4#"&$%"":
06#0&@%*%&:/*%#;&(1&06%&+,-#./-#*#&/"#0%7
a. Determining the Total Amount of pGLO plasmid DNA
A6%&0(0#"&#.()10&(=&EF>&@%&9%5#1&@406&4:&%J)#"&0(&06%&/*(;)$0&(=&06%&$(1$%10*#04(1&#1;
06%&0(0#"&<(").%&):%;?&(*
HEF>&41&K5I&&L&&H$(1$%10*#04(1&(=&EF>&41&K5-K"I&&G&&H<(").%&(=&EF>&41&K"I
M1&064:&%G/%*4.%10&'()&):%;&NO&K"&(=&/C+D&#0&$(1$%10*#04(1&(=&O7OP&K5-K"7&A64:&.%#1:&06#0
%#$6&.4$*("40%*&(=&:(")04(1&$(10#41%;&O7OP&K5&(=&/C+D&EF>7&Q#"$)"#0%&06%&!"!#$%#("'&!
",%456%):%;&41&064:&%G/%*4.%107

8"!#$%#("'&!%",%0123%456%9:-;
):%;&41&064:&%G/%*4.%10&L
TTTTTTTTTT

810%*&06#0&1).9%*&6%*%&

R(@&@4""&'()&):%&064:&/4%$%&(=&41=(*.#04(1S

44

!" #$%$&'()()*+%,$+-&./%(0)+0-+1234+15.6'(7+#89+:()+%,$+!./%$&(.;+%,.%+./%<.55=+*0%
61&$.7+0)%0+%,$+3>?.'1?.&.+15.%$@ !"#$%&#'(&)**&(+%&,-.&/'0&)11%1&('&(+%&2)$(%3")*&$%**4
5"**&2%&(3)#46%33%1&('&(+%&)7)3&8*)(%9&/'0&#%%1&('&6"#1&'0(&5+)(&63)$("'#&'6&(+%&,-.&5)4&)$(0)**/
483%)1&'#('&(+%&:;<)=8<)3)&8*)(%>&?'&1'&(+"49&1"@"1%&(+%&@'*0=%&'6&,-.&/'0&483%)1&'#&(+%
:;<)=8<)3)&8*)(%&2/&(+%&('()*&@'*0=%&'6&*"A0"1&"#&(+%&(%4(&(02%&$'#()"#"#7&(+%&,-.>&.&6'3=0*)
6'3&(+"4&4()(%=%#(&"4
U3)$("'#&'6&,-.&04%1&&S&& B'*0=%&483%)1&'#&:;<)=8&8*)(%&CD*E
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F'0&483%)1&GHH&D*&'6&$%**4&$'#()"#"#7&,-.&63'=&)&(%4(&(02%&$'#()"#"#7&)&('()*&@'*0=%&'6
IGH&D*&'6&4'*0("'#>&,'&/'0&3%=%=2%3&5+/&(+%3%&"4&IGH&D*&('()*&4'*0("'#J&:''K&"#&(+%&*)2'3)('3/
83'$%103%&)#1&*'$)(%&)**&(+%&4(%84&5+%3%&/'0&)11%1&*"A0"1&('&(+%&3%)$("'#&(02%>&.11&(+%&@'*0=%4>
L4%&(+%&)2'@%&6'3=0*)&('&$)*$0*)(%&(+%&-&./%(0)+0-+1234+15.6'(7+#89 /'0&483%)1
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?'&)#45%3&(+"4&A0%4("'#9&/'0&5"**&#%%1&('&=0*("8*/&(+%&%0%.5+.'0<)%+0-+1234+#89
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M#(%3&(+)(&#0=2%3&+%3%&
N

T+)(&5"**&(+"4&#0=2%3&(%**&/'0J

45

8Q:R&,-.&483%)1&CD7E&S
VVVVVVVV

!""#$%&$%''$(")*$+%'+)'%&,"-.$%/"012$31+,41$56,+6$"7$&61$-)8/1*.$(")$+%'+)'%&14
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;"5$).1$&61$4%&%$,-$&61$&%/'1$&"$+%'+)'%&1$&61$177,+,1-+($"7$&61$<=!>$&*%-.7"*8%&,"?*%-.7"*8%&,"-$177,+,1-+($@$$?"&%'$-)8/1*$"7$+1''.$9*"5,-9$"-$&61$%9%*$<'%&1
A8")-&$"7$3;A$.<*1%4$"-$&61$%9%*$<'%&1

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Analysis
?*%-.7"*8%&,"-$177,+,1-+($+%'+)'%&,"-.$*1.)'&$,-$01*($'%*91$-)8/1*.2$C+,1-&,.&.$"7&1-$).1
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Q%**(,-9$&6,.$,41%$%$',&&'1$7%*&61*E$.)<<".1$.+,1-&,.&.$+%'+)'%&14$%-$177,+,1-+($"7$REGGG
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46

!"#$%&"'($#)'*+(#,$-%$./011$23'"4%53*'"24678$9#3#$:'(:;('2#</$2=#"$2=#$4:&#"2&%&:$"52'2&5"
%53$2=&4$";*>#3$95;(<$>#,
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K59$95;(<$4:&#"2&424$3#+532$L01/111$23'"4%53*'"24678$&"$4:&#"2&%&:$"52'2&5"M

N#+532$D5;3$:'(:;('2#<$23'"4%53*'2&5"$#%%&:&#":D$&"$4:&#"2&%&:$"52'2&5"@

O4#$'$4#"2#":#$53$295$25$#)+('&"$9='2$D5;3$:'(:;('2&5"$5%$23'"4%53*'2&5"$#%%&:&#":D
*#'"4@

P&52#:="5(58&424$'3#$&"$8#"#3'($'83##*#"2$2='2$2=#$23'"4%53*'2&5"$+3525:5($2='2$D5;
='Q#$R;42$:5*+(#2#<$8#"#3'((D$='4$'$23'"4%53*'2&5"$#%%&:&#":D$5%$>#29##"$S@1$)$A1. '"<$H@1
)$A1G 23'"4%53*'"24$+#3$*&:3583'*$5%$TUV@
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:('44@

45+0

6..7875,89

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47

"#$%&$#'()'*()'+#,-./+0#'1/,)(..1%1(,%2)/.)'*()./$$/31,4)(56(+10(,')&-1,4)'*()1,./+0#7
'1/,)#,8)'*()+(-&$'-)$1-'(8)9($/3:

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9:3$6&$;';&9 01'.(<-1)'0*-.$(-&=0*-.
>3$6&$%?@A$%&'()*+$(-&=0*-.
9:3$6&$@B$C1-0D
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99E$,-&-.*/($-<$01'.(<-1)'.0(
;1$$)1,)'*()./$$/31,4)%*#+')#,8)-*/3)2/&+)%#$%&$#'1/,-)'/)2/&+)'(#%*(+<
G&09(+)/.)%/$/,1(-)/,)JKD#06D#+#)6$#'()P
Q1%+/4+#0-)/.)FGH)-6+(#8)/,)'*()6$#'(-)P
R+#,-./+0#'1/,)(..1%1(,%2)P

=5'+#)"+(81')"*#$$(,4(<

>.)#)6#+'1%&$#+)(56(+10(,')3(+()?,/3,)'/)*#@()#)'+#,-./+0#'1/,)(..1%1(,%2)/.)A)5)BCA
9#%'(+1#DE4)/.)FGHI)*/3)0#,2)'+#,-./+0#,')%/$/,1(-)3/&$8)9()(56(%'(8)'/)4+/3)/,)'*(
JKD#06D#+#)6$#'(L)M/&)%#,)#--&0()'*#')'*()%/,%(,'+#'1/,)/.)FGH)#,8).+#%'1/,)/.)%($$-6+(#8)/,)'*()JK)#4#+)#+()'*()-#0()#-)'*#')/.)'*()6NJO)$#9/+#'/+2:

48

Appendix A Historical Links to Biotechnology

!"#$#%"&'$()*'+,-#*.')"#+(/',(/'0('+("+)1*1,)"+%(/",)#*23(4+(56789(:*101*"&;(<*"--")/9('
=#+0#+(>/2,"&"'+(?#*;"+%("+('(>')/#$#%2($'@#*')#*29(&#+0A&)10('+(1B>1*".1+)()/')(/1
?#A$0(+1C1*(@1('@$1()#(-A$$2("+)1*>*1)(',($#+%(',(/1($"C103(<*"--")/(>1*.'+1+)$2(&/'+%10
D)*'+,-#*.10E('(,'-19(+#+>')/#%1+"&(@'&)1*"'$(,)*'"+(#-(>+1A.#&#&&A,("+)#('(01'0$2
>')/#%1+"&(,)*'"+3(F1('&&#.>$",/10()/",('.'G"+%(&/'+%1("+()/1(@'&)1*"'(@2()*1')"+%()/1(,'-1
@'&)1*"'(?")/(/1')H;"$$10(01'0$2(@'&)1*"'3(4+()/",(."B)A*1(#-()/1()?#(@'&)1*"'$(,)*'"+,()/1*1
?1*1(+#($"C"+%9(C"*A$1+)(@'&)1*"'9(@A)()/1(."B)A*1(;"$$10()/1(."&1(")(?',("+I1&)10("+)#3(F1
*1>1')10()/1(1B>1*".1+)(.'+2()".1,9('$?'2,(?")/()/1(,'.1(*1,A$),3(F1('+0(.'+2(#-(/",(
&#$$1'%A1,(?1*1(C1*2(>1*>$1B103(J/')()*'+,-#*.10(,'-1(@'&)1*"'("+)#()/1(01'0$2(;"$$1*,K
L'+2(21'*,($')1*9()/",(?#A$0(&#.1()#(@1(;+#?+(',()/1(-"*,)(*1&#*010(&',1(#-(@"#$#%"&'$
)*'+,-#*.')"#+(&#+0A&)10("+('($'@#*')#*29('+0(+#(#+1(&#A$0(1B>$'"+(")3(<*"--")/(0"0(+#)
;+#?(#-(MNO9(@A)(;+1?()/1()*'+,-#*.')"#+(?',("+/1*")'@$13(O,('+2(,"+%$1(>#"+)("+(/",)#*2
&'+(@19(<*"--")/P,(1B>1*".1+),("+()*'+,-#*.')"#+(&'+(@1(,11+(',()/1(@"*)/(#-('+'$2)"&'$(
%1+1)"&(.'+">A$')"#+()/')(/',($10()#(*1&#.@"+'+)(MNO('+0(@"#)1&/+#$#%29('+0()/1
>*#,>1&),(-#*(/A.'+(%1+1(.'+">A$')"#+3((
4+(56QQ9(,"B)11+(21'*,('-)1*(<*"--")/P,(1B>1*".1+)9('(*1,1'*&/(%*#A>(')(R#&;1-1$$1*
4+,)")A)19($10(@2(S,?'$0(T3(OC1*29(>A@$",/10('(>'>1*()/')(&'.1(0"*1&)$2(-*#.()/1(?#*;(#<*"--")/3(UJ/')(",()/1(,A@,)'+&1(*1,>#+,"@$1KV(OC1*2(?#A$0(',;(/",(&#?#*;1*,3(J#*;"+%
?")/()/1(,'.1(,)*'"+,(#-(>+1A.#+"'H&'A,"+%(@'&)1*"'9(OC1*2('+0(/",(&#?#*;1*,(>*#C"010('
*"%#*#A,('+,?1*()#()/')(WA1,)"#+3(T/12(>*#C10()/')()/1(,A@,)'+&1(",(MNO9('+0()/')(@"#$#%"&'$
)*'+,-#*.')"#+(",(>*#0A&10(?/1+(&1$$,()';1(A>('+0(1B>*1,,(-#*1"%+(MNO3(O$)/#A%/(")()##;
.'+2(21'*,(-#*(&*10")()#(@1(%"C1+()#(OC1*29()#0'2(/1(",(A+"C1*,'$$2('&;+#?$10%10(-#*()/",
-A+0'.1+)'$('0C'+&1("+(@"#$#%"&'$(;+#?$10%13(!A"$0"+%(A>#+()/1(?#*;(#-(OC1*2('+0(
#)/1*,9(M#A%$',(F'+'/'+(01C1$#>10()/1()1&/+"WA1(#-(&#$#+2()*'+,-#*.')"#+(A,10("+()/",
"+C1,)"%')"#+359(7
Historical Context
!"#"$%&'()*#+,-).*$%-#
58XYZ<*1%#*([#/'++(L1+01$\(L1+01$(>*1,1+)10(/",(-"+0"+%,(01,&*"@"+%()/1(>*"+&">$1,(@2
?/"&/(%1+1)"&()*'"),('*1(>',,10(-*#.(>'*1+)()#(#--,>*"+%3(:*#.(/",(?#*;()/1(&#+&1>)(#)/1(%1+1(',()/1(@',"&(A+")(#-(/1*10")2(?',(01*"C103((
56]]Z^'*$(^#**1+,9(FA%#(M1(_*"1,9(`*"&/(T,&/1*.';\(a$'+)(%1+1)"&",),(&#+0A&)"+%(
"+/1*")'+&1(,)A0"1,(A+&#C1*10()/')()/1"*(?#*;(?',(1,,1+)"'$$2('(0A>$"&')1(#-(?#*;(
>1*-#*.10(+1'*$2(-#A*(01&'01,(1'*$"1*(@2('+(A+;+#?+(OA,)*"'+(OA%A,)"+"'+(.#+;9
<*1%#*([#/'++(L1+01$9(?/#(,)A0"10(>1',3
5678Z:*101*"&;(<*"--")/\(<*"--")/()*'+,-#*.10(+#+>')/#%1+"&(/%01-&-&&2+'0#"2.-#%* "+)#
>')/#%1+"&(@'&)1*"'(A,"+%(/1')H;"$$10(C"*A$1+)(@'&)1*"'3(F1(,A%%1,)10()/')()/1()*'+,-#*."+%
-'&)#*(/'0(,#.1)/"+%()#(0#(?")/()/1(>#$2,'&&/'*"01(&'>,A$1(,2+)/1,",3(<*"--")/(0"0(+#)
;+#?(#-(MNO9(@A)(;+1?()/1()*'+,-#*.')"#+(?',("+/1*")'@$13(<*"--")/P,(1B>1*".1+),("+
)*'+,-#*.')"#+(&'+(@1(,11+(',()/1(@"*)/(#-('+'$2)"&'$(%1+1)"&(.'+">A$')"#+()/')(/',($10()#
*1&#.@"+'+)(MNO()1&/+#$#%2('+0()/1(>*#,>1&),(-#*(/A.'+(%1+1(.'+">A$')"#+3
56QQZS,?'$0(OC1*29(^#$"+(L'&=1#0\((OC1*2('+0(/",(&#$$1'%A1,('++#A+&10()/')()/12(/'0
",#$')10()/1()*'+,-#*."+%(-'&)#*()#('(/"%/(>A*")29('+0(")(?',(MNO3(b"+&1()/",(&$',,"&(
1B>1*".1+)("+(.#$1&A$'*(%1+1)"&,9()*'+,-#*.')"#+9(&#+IA%')"#+(D@'&)1*"'$(.')"+%E9('+0
)*'+,0A&)"#+(DC"*'$(MNO()*'+,-1*E(/'C1(@11+(A,10()#()*'+,-1*(%1+1,(@1)?11+(,>1&"1,(#@'&)1*"'9(/)-+-03%1*9(."&19(>$'+),('+0('+".'$,9(.'..'$"'+(&1$$,("+(&A$)A*19('+0(-#*
/A.'+(%1+1()/1*'>23((

49

!"#$%&'()*+,-*)./*01,23)4/3,5/3.*6,-*)./*0,37+,5/3.*,8.*+,)3+9:9.:4:;*.,:(,.8'(8),37+
;/:.;/:)8.1,37+,<3=4*)9:;/3>*,?$,4:,./:@,=:7='8.9A*'0,4/34,BC&,@3.,4/*,97(:)D349:7
D:'*=8'*,:(,/*)*+940E,&':7>,@94/,4/*,@:)F,:(,&A*)01,23=G*:+1,37+,2=53)401,4/*
-*)./*0H5/3.*,*I;*)9D*74,.*3'*+,4/*,87+*).437+97>,4/34,BC&,@3.,4/*,4)37.(:)D97>
D34*)93',37+,4/*,97(:)D349:7,D:'*=8'*,:(,/*)*+940E
!"J$%K38',L*)>1,M37*4,2*)4N6,L*)>,8.*+,4/*,7*@'0,+9.=:A*)*+,*7+:78='*3.*,*7N0D*1,!"#OP1
4:,=84,QRST,BC&,37+,<3=4*)9:;/3>*,K$$,BC&1,37+,4/*7,8.*+,4*)D973',4)37.(*)3.*
*7N0D*,37+,BC&,'9>3.*,4:,)*U:97,4/*.*,.*;3)34*,;9*=*.,974:,:7*,;9*=*,:(,BC&E
5)*349:7,:(,4/*,(9).4,)*=:D<97374,BC&,D:'*=8'*,@3.,4/*,<*>97797>,:(,4/*,3>*,:(
<9:4*=/7:':>0E,?/*,7*@,D:'*=8'*,@3.,7:4,;'3=*+,97.9+*,3,D3DD3'937,=*'',<*=38.*,:(
=:7=*)7.,97,4/*,.=9*749(9=,=:DD87940,)*>3)+97>,>*7*49=,4)37.(*).E
!"JV%-*)<*)4,L:0*)1,Q437'*0,5:/*71,&779*,5/37>6,L*)>1,L:0*)1,37+,5:/*7,8.*+
!"#OP,4:,9.:'34*,37,9743=4,>*7*,(:),F373D0=97,)*.9.437=*E,L:0*)1,5:/*71,37+
5/37>,.;'9=*+,4/*,F373D0=97,)*.9.437=*,>*7*,974:,37,!"#OP,=84,;'3.D9+,4/34
3')*3+0,=:74397*+,4*4)3=0='97*,)*.9.437=*,37+,;):+8=*+,3,)*=:D<97374,<3=4*)93'
;'3.D9+,D:'*=8'*,@94/,+83',3749<9:49=,)*.9.437=*E,?/*0,4/*7,4)37.(:)D*+ !$%"#&'
@94/,4/9.,*7>97**)*+,;'3.D9+E
!"JJ%W*7*74*=/1,P7=E6,?/*,(9).4,;):+8=4,:(,>*7*49=,*7>97**)97>1,4/*,>*7*,(:),/8D37,
.:D34:.43497,X/8D37,>):@4/,/:)D:7*Y)*'*3.97>,97/9<94:)0,(3=4:)Z1,@3.,*I;)*..*+,97,
<3=4*)93,37+,377:87=*+,<0,W*7*74*=/E
!"[T%ME,\E,W:)+:71,])37F,O8++'*6,W:)+:7,37+,O8++'*,.8==*..(8''0,D9=):97U*=4*+,7:)D3'
>*7*.,974:,D:8.*,>*)DY'97*,=*''.E
!"[$%O9=/3)+,K3'D94*)1,O3';/,L)97.4*)6,K3'D94*),37+,L)97.4*),D9=):97U*=4*+,4/*,>*7*,(:)
)34,>):@4/,/:)D:7*,974:,D9=*,*D<)0:.E,?/9.,@3.,4/*,(9).4,>*7*49=,>*)DY'97*,^=8)*_,)*;:)4*+
97,3,D3DD3'E,?/*,)*=9;9*74,D:8.*,@3.,=3''*+,^'944'*_,<*=38.*,94,.8((*)*+,():D,3,(:)D
:(,=:7>*7943',+@3)(9.DE
!"[[%Q4*A*7,O:.*7<*)>6,O:.*7<*)>,37+,/9.,=:''*3>8*.,@*)*,>9A*7,3;;):A3',4:,;*)(:)D
4/*,(9).4,>*7*,4)37.(*),*I;*)9D*74,97,/8D37,;349*74.,.8((*)97>,():D,D*43.4349=
D*'37:D3E,?/9.,*I;*)9D*74,)*;)*.*74*+,>*7*49=,4)3=F97>,@94/,4/*,D3)F*),>*7*,C*:O
37+,7:4,>*7*,4/*)3;0E,
!""T%\E,])*7=/,&7+*).:71,29=/3*',L'3*.*1,`*77*4/,58'A*)6,&4,!$6#$,;EDE,:7,])9+301
Q*;4*D<*),!S1,!""T,34,4/*,C349:73',537=*),P7.49484*1,3,(:8),0*3),:'+,>9)'1,&./374/9,B*
Q9'A3,():D,5'*A*'37+1,a/9:1,<*=3D*,4/*,(9).4,/8D37,>*7*Y4/*)3;0,;349*74E,Q/*,@3.
97(8.*+,@94/,/*),:@7,@/94*,<'::+,=*''.,=3))097>,4/*,=:))*=4*+,A*).9:7,:(,4/*,3+*7:.97*
+*3D973.*,X&B&Z,>*7*E,B).E,&7+*).:71,L'3*.*,37+,58'A*),+9+,7:4,*I;*=4,D*3797>(8'
)*.8'4.,():D,4/*,*I;*)9D*74,(:),3<:84,!,0*3)E,&,.*=:7+,>9)'1,5074/93,584./3''1,@3.,
.9D9'3)'0,97U*=4*+,97,!""TE,O*;:)4.,97,M87*,!""V,./:@*+,4/*,4@:,>9)'.,@94/,.D9'*.,37+
=/9'+9./,*7*)>01,;'3097>,97,3,.=/::',03)+E,L:4/,>9)'.b,9DD87*,.0.4*D.,@*)*,@:)F97>
*((*=49A*'0E
!""S%a4/*),>*7*,4/*)3;0,=37+9+34*.,97='8+*,.9=F'*,=*'',37*D931,/*D:;/9'931,+93<*4*.1,
=37=*)1,37+,/*3)4,+9.*3.*,;349*74.E,W*)D,'97*,>*7*,4/*)3;0,9.,+*<34*+,+8)97>,D**497>,:(
4/*,O*=:D<97374,BC&,&+A9.:)0,5:DD944**E,L0,!""c,3,>):@97>,78D<*),:(,;):;:.3'.
3@394,)*A9*@,<0,4/*,-8D37,W*7*,?/*)3;0,Q8<=:DD944**,:(,4/*,O*=:D<97374,BC&
&+A9.:)0,5:DD944**E
!""#%G*+,<0,ME,5)39>,R*74*)1,3,>):8;,34,4/*,97.49484*,(:),W*7:D9=,O*.*3)=/,X?PWOZ,97
23)0'37+1,;8<'9./*+,4/*,(8'',>*7*,.*d8*7=*,:(,4/*,<3=4*)98D,()*#+,'&-.%'/0&-)/12)3 3
'37+D3)F,97,D9=):<9:':>9=3',)*.*3)=/,3.,4/*,(9).4,()**Y'9A97>,:)>379.D,@/:.*,>*7*49=
^<'8*;)974_,@3.,+*=:+*+E,,
!""c%&,D8'497349:73',=:''3<:)349:7,97='8+97>,D:)*,4/37,!TT,'3<:)34:)9*.,():D,e8):;*1

50

!"#$%&'(')'$%'()%*'+'(%,'-%./0%123-.%.4%5(3'607%./0%0(.230%80(490%-0:50(;0%41%'
05<'3=4.0$%./0%=0'-.%!"##$"%&'(#)*+#)%),-*-")>%!.+#)%),-*-") 2-%'%;49903;2'77=%-28(212?
;'(.%=0'-.%;4994(7=%5-0)%2(%@'<2(8%'()%2(%10390(.'.24(%41%'7;4/472;%@0603'80-%'()%2,2)07=%5-0)%2(%./0%7'@43'.43=%'-%'%94)07%438'(2-9%143%5()03-.'()2(8%;07757'3%'()
9470;57'3%+34;0--0-%41%05<'3=4.0->
ABBCD";20(.2-.-%70)%@=%E'(%F2795.%'.%";4.7'()G-%H4-72(%E(-.2.5.0%30+43.0)%./0%-5;;0--157
;74(2(8%41%'%-/00+$%('90)%I477=$%1349%./0%;077%41%'(%')57.%5))03%;077>%J/0%;74(2(8%41
I477=%-+'3<0)%2(.03('.24('7%)0@'.0%'@45.%0./2;'7%'()%943'7%2--50-%;4(;03(2(8%;74(2(8>
"5@-0:50(.7=$%-;20(.2-.-%'.%";4.7'()G-%H4-72(%E(-.2.5.0$%2(%;477'@43'.24(%,2./%";4.7'()?
@'-0)%KKL%J/03'+05.2;-$%-5;;0--1577=%;74(0)%.,4%80(0.2;'77=%94)2120)%7'9@-$%('90)
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5-0)%2(%.30'.2(8%/59'(%/094+/272'>
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TU$UUU%<(4,(%/59'(%80(0-$%'%9270-.4(0%143%./0%V59'(%W0(490%K34X0;.>%%
YUUUD#%.0'9%70)%@=%E(84%K4.3=<5-%41%./0%",2--%Z0)03'7%E(-.2.5.0%41%J0;/(4748=%2(%[532;/
'()%K0.03%\0=03%41%./0%!(2603-2.=%41%Z302@538%2(%W039'(=%30+43.0)%./0%;30'.24(%41
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'%;477'@43'.24(%@0.,00(%'%+326'.0%;49+'(=$%&0703'%W0(492;-$%'()%30-0'3;/03-%,437)?
,2)0%-.5)=2(8%./0%1352.%17=>%%6.+')4"0&5"*3)%$%'%94)07%5-0)%,2)07=%5-0)%2(%./0%7'@43'.43=$
2-%./0%7'380-.%'(29'7%-4%1'3%.4%/'60%2.-%80(0.2;%;4)0%)0;2+/030)>%%
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&0703'%W0(492;-%'7-4%'((45(;0)%;49+70.24(%41%./023%a123-.%'--09@7=G%41%./0%80(490>%%

51

!""#$%&'()*+,-+.'#!/'!""#/'0)1)+-'2)&34567'-&8'9:)';&9)+&-953&-1'<,4-&'2)&34)
=)>,)&65&?'03&73+95,4'@35&91.'-&&3,&6)8'9:)'A,*157:5&?'3B'9:)'&)-+1.'634A1)9)
7)>,)&6)'3B'9:)':,4-&'?)&34)'C'9:)'?)&)956'D*1,)A+5&9E'B3+'-':,4-&'*)5&?F'G:57
-6634A157:4)&9'933H'9:)'5&9)+&-953&-1'9)-4'-14379'9I)&9.'.)-+7'-&8'5&J31J)8'9:)'
6311-*3+-953&'3B'9:3,7-&87'3B'765)&95797'B+34'-+3,&8'9:)'I3+18F'0)1)+-'2)&34567
+)A3+9)8'634A1)95&?'9:)'I3+H'5&'-AA+3K54-9)1.'&5&)'43&9:7F'G:)'9I3'?+3,A7'85BB)+)8
5&'9:)5+')7954-9)7'B3+'9:)'&,4*)+'3B'?)&)7'5&'9:)':,4-&'?)&34)/'*,9'9:)'+-&?)'A+)8569C
)8'*.'*39:'?+3,A7/'*)9I))&'!L/"""'-&8'M"/"""'?)&)7/'57'B-+'B)I)+'9:-&'9:)'A+)J53,7
)7954-9)'3B'#""/"""'?)&)7F'G:57',&)KA)69)8'B5&85&?'7,??)79)8'9:-9'-&'3+?-&574'-7
634A1)K'-7'-':,4-&'*)5&?'6-&'*)'4-8)'3B'73'B)I'?)&)7/'3&1.'9I56)'-7'4-&.'-7'B3,&8
5&'9:)'I3+4'!F'"#"$%&' 3+'9:)'B1.'()*+"#%&,$%'-".F'G:)',&J)515&?'3B'9:)'B,11'7)>,)&6)
3B'9:)':,4-&'?)&34)'4-H)7'59'A3775*1)'B3+'+)7)-+6:)+7'-11'3J)+'9:)'I3+18'93'*)?5&
8)J)13A5&?'9+)-94)&97'B3+'4-&.'857)-7)7F'
N+)758)&9'2)3+?)'O,7:'8)658)8'9:-9'3&1.')KA)+54)&97'5&J31J5&?'9:)')K5795&?'PM')4*+.C
3&56'79)4'6)11'15&)7'I3,18'*)')15?5*1)'B3+'A3775*1)'B)8)+-1'B,&85&?F'G:)'A+)758)&9Q7
8)65753&'I-7'857-AA35&95&?'93'4-&.'765)&95797'I:3':3A)8'93',7)')4*+.3&56'79)4'6)117
93'8)J)13A'9+)-94)&97'B3+'4-&.'-514)&97F
R8J-&6)8'0)11'G)6:&313?./'-'74-11'634A-&.'5&'S-77-6:,7)997/'-&&3,&6)8'9:-9'59':-8
7,66)77B,11.'613&)8':,4-&')4*+.37'B3+'9:)'A,+A37)'3B')K9+-695&?'9:)5+'79)4'6)117F'G:57
4)9:38'63,18',1954-9)1.'*)',7)8'93'9+)-9'A-95)&97'I59:'-'J-+5)9.'3B'857)-7)7'*.'4-H5&?
+)A1-6)4)&9'6)117/'7,6:'-7'&)+J)'-&8'4,761)'6)117/'I:56:'6-&'*)'9+-&7A1-&9)8'*-6H'5&93
7-4)'A)+73&'I59:3,9'9:)'+57H'3B'*)5&?'+)@)69)8'*.'9:)'*38.F''
NNT'G:)+-A),9567/'9:)'634A-&.'9:-9':)1A)8'93'613&)'U311.'9:)'7:))A/'-&&3,&6)8'9:-9'59
:-8'613&)8'B5J)'?)&)956-11.'4385B5)8'A5?1)97'I59:'-&'5&-695J-9)8/'3+'DH&36H)8'3,9E/
?)&)'9:-9'I3,18'4-H)'9:)5+'3+?-&7'4,6:'1)77'15H)1.'93'*)'+)@)69)8'I:)&'9+-&7A1-&9)8
5&93'-':,4-&'+)65A5)&9F'G:)'7,66)77'3B'NNT'G:)+-A),9567'*+5&?7':3A)'93'9:)'9:3,7-&87
3B'A)3A1)'I:3'-+)'I-595&?'93'+)6)5J)'83&-9)8'3+?-&7'7,6:'-7':)-+97/'1,&?7/'H58&).7/'-&8
15J)+7F''
!""!$U311.'9:)'7:))A/'9:)'B5+79'4-44-1'93'*)'613&)8'B+34'-&'-8,19'6)11/'8)J)13A)8'-+9:+5C
957'-9'-'+)1-95J)1.')-+1.'-?)'3B'B5J)'.)-+7F';9'57'&39'61)-+'I:)9:)+'U311.Q7'63&85953&'I-7
9:)'+)7,19'3B'-'?)&)956'8)B)69'6-,7)8'*.'613&5&?/'3+'I:)9:)+'59'I-7'-'4)+)'635&658)&6)F
G:)'&)I7':-7'+)&)I)8'8)*-9)7'3&'I:)9:)+'613&)8'-&54-17'-+)'7,76)A95*1)'93'A+)4-9,+)
-?5&?'-&8':)-19:'A+3*1)47'-&8':-7'-173'*))&'-'7)9*-6H'B3+'9:37)'I:3'-+?,)'9:-9'613&5&?
6-&'*)',7)8'93'?)&)+-9)'-'7,AA1.'3B'3+?-&7'93':)1A'A-95)&97'3&'9:)'9+-&7A1-&9'1579F''

52

Appendix B Glossary of Terms

!"#$

!"#$%&'()*)(#+%&),*-"%.)-#)(/00#"-)1*2-'"%*+)3"#4-56
7#8-*%8()8/-"%'8-),%.-/"')#9)2*"1#5:&"*-'(;)*,%8#
*2%&(;)8/2+'#-%&'(;)(*+-(;)*8&)$%-*,%8(6

!%&'(')&'*+,-.-*&')%

<(')#9)*8)*8-%1%#-%2)-#)('+'2-)1*2-'"%*)2#8-*%8%83)-5')=>?
#9)%8-'"'(-6)@5')0ABC)0+*(,%&)=>?)2#8-*%8()-5')3'8')9#"
1'-*D+*2-*,*(')-5*-)0"#$%&'()"'(%(-*82')-#)-5')*8-%1%#-%2
*,0%2%++%86)C82')1*2-'"%*)*"')-"*8(9#",'&)4%-5)-5')0ABC
0+*(,%&;)-5':)1'3%8)0"#&/2%83)*8&)('2"'-%83)1'-*D+*2-*,*('
0"#-'%86)E'2"'-'&)1'-*D+*2-*,*(')1"'*F()&#48)*,0%2%++%8;
"'8&'"%83)-5')*8-%1%#-%2)5*",+'(()-#)-5')1*2-'"%*+)5#(-6)C8+:
1*2-'"%*)2#8-*%8%83)-5')0ABC)0+*(,%&)2*8)3"#4)*8&)9#",
2#+#8%'()%8)8/-"%'8-),'&%/,)2#8-*%8%83)*,0%2%++%8;)45%+'
/8-"*8(9#",'&)2'++()-5*-)5*$')8#-)-*F'8)/0)-5')0ABC)
0+*(,%&)2*88#-)3"#4)#8)-5')*,0%2%++%8)('+'2-%#8)0+*-'(6

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-5')*8-%1%#-%2)*,0%2%++%86)@5')1'-*D+*2-*,*(')0"#-'%8)%()
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-"*8(9#",'& 4%-5)*)0+*(,%&)2#8-*%8%83)-5')3'8')9#")
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-5')*,0%2%++%8)0"'('8-)%8)-5')BG)8/-"%'8-)*3*";)45%25
*++#4()9#")1*2-'"%*+)3"#4-5)*8&)'.0"'((%#8)#9)8'4+:
*2H/%"'&)3'8'()*+(#)2#8-*%8'&)#8)-5')0+*(,%&;)!"#"$ AI!6

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67.&7$-+8-9'#

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*9-'")B/"%*)*8&)G'"-*8%K)1"#-5)*8&)*3*")*"'),*&')9"#,
*8)'.-"*2-)#9):'*(-)*8&)*8)'8L:,*-%2)&%3'(-)#9),'*1:0"#&/2-( 45%25)0"#$%&')*),%.-/"')#9)2*"1#5:&"*-'(;
*,%8#)*2%&(;)8/2+'#-%&'(;)(*+-(;)*8&)$%-*,%8(;)*++)#9
45%25)*"')8/-"%'8-()9#")1*2-'"%*+)3"#4-56)?3*";)45%25)%(
9"#,)('*4''&;)0#+:,'"%L'()45'8)5'*-'&)*8&)2##+'&)-#
9#",)*)(#+%&)3'+)J(%,%+*")-#)M'++DC)3'+*-%8K;)*8&)
9/82-%#8()-#)0"#$%&')*)(#+%&)(/00#"-)#8)45%25)-#)2/+-/"'
-5')1*2-'"%*6

53

!"#"$%&'(#)%#""*%#)

!"#$%&'()*+&,(-'$-.$&'$-/0&'(1%21$0#'#,(3$%&,#/(&+
45678$9:$(',/-;*3('0$-/$#+(%('&,('0$1)#3(.(3$0#'#1<

!"#"'+"),-.$%/#

=#'#$#>)/#11(-'$('$&++$-/0&'(1%1$(1$3&/#.*++:$/#0*+&,#;
,-$&++-?$.-/$;(..#/('0$3-';(,(-'1$&';$,-$)/#@#',$?&1,#.*+
-@#/)/-;*3,(-'$-.$*''##;#;$)/-,#('1<$!"#$0#'#1
('@-+@#;$('$,"#$,/&'1)-/,$&';$9/#&A;-?'$-.$.--;$&/#
0--;$#>&%)+#1$-.$"(0"+:$/#0*+&,#;$0#'#1<$B-/$#>&%)+#C
,"#$1(%)+#$1*0&/C$&/&9('-1#C$3&'$9#$*1#;$&1$&$1-*/3#$-.
#'#/0:$&';$3&/9-'$9:$9&3,#/(&<$!"#$9&3,#/(&+$#'D:%#1
,"&,$&/#$'##;#;$,-$9/#&A$;-?'$-/$;(0#1,$&/&9('-1#$.-/
.--;$&/#$'-,$#>)/#11#;$('$,"#$&91#'3#$-.$&/&9('-1#$9*,
&/#$#>)/#11#;$?"#'$&/&9('-1#$(1$)/#1#',$('$,"#
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,*/'#;$-'<$F"#'$&/&9('-1#$/*'1$-*,$,"#1#$0#'#1$&/#
,*/'#;$9&3A$-..<$G##$7))#';(>$5$.-/$&$%-/#$;#,&(+#;
#>)+&'&,(-'$-.$,"#$/-+#$,"&,$&/&9('-1#$)+&:1$('$,"#
/#0*+&,(-'$&';$#>)/#11(-'$-.$,"#$=/##'$B+*-/#13#',
H/-,#('$0#'#<

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=/##'$B+*-/#13#',$H/-,#('$4=BH8$?&1$-/(0('&++:$(1-+&,#;
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-..$#'#/0: ('$,"#$.-/%$-.$@(1(9+#$0/##'$+(0",<$$

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3&//:('0$-'#$-/$%-/#$0#'#1$.-/$&',(9(-,(3$/#1(1,&'3#
)/-,#('1$&';$&$3+-'#;$.-/#(0'$0#'#$1*3"$&1$=BH<

5!67

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&';$.*'3,(-'<

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+(9/&/:<

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3-',&%('&,(-'$;*/('0$&'$#>)#/(%#',$,"/-*0"$
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-'$(,$&3/-11$&'$&0&/$)+&,#

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('1#/,#;$&';$,"#'$)/-)&0&,#;$('$&$"-1,$3#++<

54

Appendix C Basic Molecular Biology Concepts and

Terminology
!"#$%&'"()"$*+",-.-/0"1(2,&"2+.+3,#"$*3$"3,,",-.-/0"(203/-#4#"(203/-5+"$*+4#+,.+#"-/
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32+"(203/-5+&"$("9+2)(24"-/";(/;+2$7
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:+,,#";3/"8+"03$*+2+&")2(4"$*+-2"/3$%23,",(;3$-(/#"3/&"02(1/"-/#-&+",38(23$(2'";(/$3-/+2#7
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'+3#$"32+".+2'"+3#'"$("02(1"-/";%,$%2+7":+,,#"$3@+/")2(4"9,3/$#="-/#+;$#"3/&"3/-43,#";3/"3,#(
8+"02(1/="8%$"32+"4(2+"&-))-;%,$"$(";32+")(27
!)$+2"02(1$*"-#";(49,+$+=";+,,#"-/";%,$%2+";3/"8+"*32.+#$+&"3/&"#$%&-+&7
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9(9%,3$-(/"1-,,"8+"0+/+$-;3,,'"-&+/$-;3,7"B%;*"3"9(9%,3$-(/"-#";3,,+&";,(/3,7">*+"92(;+##"()
;2+3$-/0"3";,(/3,"9(9%,3$-(/"-#";3,,+&";,(/-/07">*+"9%29(#+"()"#$2+3@-/0"83;$+2-3"(/"3032"-#
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()"$*+";+,,7
>("#$%&'"$*+#+"4(,+;%,+#"1+"92+932+"3";,(/3,"9(9%,3$-(/")2(4"3";+,,"$'9+"()"-/$+2+#$=
82+3@"(9+/"$*+";+,,#"3/&"#(2$"$*+";(/$+/$#7"C(2"-/#$3/;+="-$"-#")3-2,'"+3#'"$("#+9323$+"3,,"$*+"
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$'9+"-#"3,#("9(##-8,+7"J3;*"$'9+"()"92($+-/"*3#"%/-6%+"9*'#-;3,"3/&";*+4-;3,"92(9+2$-+#7
>*+#+"92(9+2$-+#"3,,(1"$*+"#+9323$-(/"()"92($+-/"#9+;-+#"83#+&"(/"#-5+=";*320+="(2
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A+"1-,,"$3@+"3";,(#+",((@"3$"$*2++".+2'"#9+;-3,"@-/&#"()"4(,+;%,+#")(%/&"-/#-&+";+,,#K
DE!="LE!"3/&"92($+-/#7"J3;*"()"$*+#+"4(,+;%,+#"9+2)(24#"3"&-))+2+/$")%/;$-(/7"DE!
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+I+;%$+"$*+"-/#$2%;$-(/#"1*-;*"32+"#$(2+&"-/"DE!7"H2($+-/#"32+"&+#-0/+&"$("9+2)(24
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<=>?@2"%/(.3"+$'#%0"87#'0"%;,+%).,#,*.&'#%.(;,+8'0.,(
>*+"43#$+2"#;2-9$")(2"+3;*"(203/-#4"-#"+/;(&+&"1-$*-/"-$#"&+(I'2-8(/%;,+-;"3;-&
FDE!G7">*+"-/)(243$-(/"1-$*-/"$*+"DE!"4(,+;%,+M#"()"+3;*";+,,"-#"#%))-;-+/$"$("-/-$-3$+
+.+2'")%/;$-(/"$*3$";+,,"1-,,"9+2)(247
DE!"4(,+;%,+#"32+".+2'",(/0";*3-/#";(49(#+&"()"2+9+3$-/0"#%8%/-$#7"J3;*"#%8%/-$
F/%;,+($-&+G";(/$3-/#"(/+"()")(%2"9(##-8,+"83#+#"92($2%&-/0")2(4"-$#"#-&+K
3&+/-/+"F>G"
$*'4-/+"F@G

;'$(#-/+"F!G
0%3/-/+"FAG

55

!"#$%&#'$(%)*"+%,&-.%&/)"#%+&0%-+1*)1*-"(2&-&()#3&,*.-#+&)4&567&%,,%#*"-((8&$)#,",*,&)4&$0%9"$-(&:-$;:)#%&<"*0&:-,%,&=.)*.'+"#3&-()#3&"*,&,"+%>&?0%&"#4).9-*")#&$-.."%+&:8&*0",
9)(%$'(%&",&%#$)+%+&"#&*0%&,%@'%#$%&)4&*0%&:-,%,&!2&"2&#2&-#+&$ -()#3&"*,&(%#3*0>
%&'()*+,-.(,)/&01-2)-&)1&-()34&+-)56!)2-,+7-+,(
A>&& B%$-',%&*0%&,':'#"*,&)4&567&$0-"#,&-.%&/)"#%+&0%-+1*)1*-"(2&*0%&,%@'%#$%&",&+".%$*")#-(
"$#!!>&B8&$)#C%#*")#2&<%&<."*%&567&,%@'%#$%&4.)9&*0%&4.%%&DE&%#+&)4&*0%&:-$;:)#%
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2"*&";90)=$&)/20,=#+*=&)/2,&9'+2*0)':&+*::$&9;&20'/$-,0.'2),/%&)2&)$&*'$;&2,&$*:*+2&2",$*&
9'+2*0)'&2"'2&"'F*&0*+*)F*=&2"*&(:'$.)=J9*+'#$*&2"*;&"'F*&'+O#)0*=&2"*&'9):)2;&2,&10,6&)/
2"*&(0*$*/+*&,-&2"*&'/2)9),2)+%&6"*0*'$&'::&,2"*0&9'+2*0)':&+*::$&'0*&C)::*=>
012("&3)#)&*$
I"*/&345&)$&*A20'+2*=&-0,.&'&1)F*/&+*::&2;(*%&)2&+'/&9*&+#2&)/2,&()*+*$&'/=&2"*&()*+*$
+'/&9*&+:,/*=&*/&.'$$*&)/2,&'&(,(#:'2),/&,-&(:'$.)=$>&!")$&(0,+*$$&(0,=#+*$&'&(,(#:'2),/
,-&";90)=&P0*+,.9)/'/2Q&345$>&5-2*0&)/20,=#+)/1&2"*$*&";90)=$&9'+C&)/2,&+*::$%&*'+"
20'/$-,0.*=&+*::&6)::&"'F*&0*+*)F*=&'/=&(0,('1'2*=&,/*&#/)O#*&";90)=>&KF*0;&";90)=&6)::
+,/2')/&2"*&$'.*&F*+2,0&345&9#2&'&=)--*0*/2&)/$*02&345>
8-&2"*0*&'0*&R%SSS&=)--*0*/2&345&.,:*+#:*$&)/&2"*&,0)1)/':&.)A2#0*%&R%SSS&=)--*0*/2
";90)=$&6)::&9*&-,0.*=L&R%SSS&=)--*0*/2&20'/$-,0.'/2&+*::$&6)::&9*&0*+,F*0*=%&*'+"&+'00;)/1
,/*&,-&2"*&,0)1)/':&R%SSS&()*+*$&,-&1*/*2)+&)/-,0.'2),/>&N#+"&'&+,::*+2),/&)$&+'::*=&'&345
:)90'0;>&8-&2"*&,0)1)/':&*A20'+2&+'.*&-0,.&"#.'/&+*::$%&2"*&:)90'0;&)$&'&"#.'/&:)90'0;>
8/=)F)=#':&345$&,-&)/2*0*$2&+'/&9*&-)$"*=&,#2&,-&$#+"&'&:)90'0;&9;&$+0**/)/1&2"*&:)90'0;
6)2"&'/&'((0,(0)'2*&(0,9*>

59

Appendix D Gene Regulation

!"#$%&'()*$+&,-.(,$-/&"*.,'*$&0$'(00)#),-$1#&-)(,*$2/(+/$1)#0&#3$3.,4$'(00)#),-$5&%*6
7(8)*-(9)$),:43)*$.#)$1#&-)(,*;$*&3)$&0$-/)$/&#3&,)$*(8,.<*$-/.-$#",$-/#&"8/$&"#$%&'()*
.,'$-/)$.,-(%&'()*$1#&-)+-(,8$"*$0#&3$'(*).*)$.#)$1#&-)(,*6$=/)$(,0&#3.-(&,$0&#$.**)3%<(,8
.$1#&-)(,$(*$+.##()'$(,$&"#$7>?6$=/)$*)+-(&,$&0$7>?$2/(+/$+&,-.(,*$-/)$+&')$0&#$3.@(,8$.
1#&-)(,$(*$+.<<)'$.$8),)6$=/)#)$.#)$&9)#$ABCBBBDEBBCBBB$8),)*$(,$-/)$/"3.,$8),&3)6$F.+/
8),)$+&')*$0&#$.$",(G")$1#&-)(,H$&,)$8),)C$&,)$1#&-)(,6$=/)$8),)$-/.-$+&')*$0&#$.$'(8)*-(9)
),:43)$(,$4&"#$3&"-/$(*$'(00)#),-$0#&3$&,)$-/.-$+&')*$0&#$.,$.,-(%&'4$&#$-/)$1(83),-$-/.+&<&#*$4&"#$)4)*6$$
!#8.,(*3*$#)8"<.-)$)I1#)**(&,$&0$-/)(#$8),)*$.,'$"<-(3.-)<4$-/)$.3&",-*$.,'$@(,'*$&0
1#&-)(,*$1#)*),-$2(-/(,$-/)(#$+)<<*$0&#$.$34#(.'$&0$#).*&,*C$(,+<"'(,8$')9)<&13),-.<$+/.,8)*C
+)<<"<.#$*1)+(.<(:.-(&,C$.,'$.'.1-.-(&,$-&$-/)$),9(#&,3),-6$J),)$#)8"<.-(&,$,&-$&,<4$.<<&2*$0&#
.'.1-.-(&,$-&$'(00)#(,8$+&,'(-(&,*C$%"-$.<*&$1#)9),-*$2.*-)0"<$&9)#1#&'"+-(&,$&0$",,))')'
1#&-)(,*$2/(+/$2&"<'$1"-$-/)$&#8.,(*3$.-$.$+&31)-(-(9)$'(*.'9.,-.8)6$=/)$8),)*$(,9&<9)'$(,
-/)$-#.,*1&#-$.,'$%#).@'&2,$K+.-.%&<(*3L$&0$0&&'$.#)$8&&'$)I.31<)*$&0$/(8/<4$#)8"<.-)'
8),)*6$M&#$)I.31<)C$-/)$*"8.#$.#.%(,&*)$(*$%&-/$.$*&"#+)$&0$),)#84$.,'$.$*&"#+)$&0$+.#%&,6$!"
#$%&'%.+-)#(.$1#&'"+)$-/#))$),:43)*$K1#&-)(,*L$,))')'$-&$'(8)*-$.#.%(,&*)$.*$.$0&&'$*&"#+)6
=/)$8),)*$2/(+/$+&')$0&#$-/)*)$),:43)*$.#)$,&-$)I1#)**)'$2/),$.#.%(,&*)$(*$.%*),-C$%"-$-/)4
.#)$)I1#)**)'$2/),$.#.%(,&*)$(*$1#)*),-$(,$-/)(#$),9(#&,3),-6$N&2$(*$-/(*$*&O
P)8"<.-(&,$&0$-/)$)I1#)**(&,$&0$1#&-)(,*$&0-),$&++"#*$.-$-/)$<)9)<$&0$-#.,*+#(1-(&,$0#&3
7>?$(,-&$P>?6$=/(*$#)8"<.-(&,$-.@)*$1<.+)$.-$.$9)#4$*1)+(0(+$<&+.-(&,$&,$-/)$7>?$-)31<.-)C
+.<<)'$.$1#&3&-)#C$2/)#)$P>?$1&<43)#.*)$*(-*$'&2,$&,$-/)$7>?$.,'$%)8(,*$-#.,*+#(1-(&,$&0
-/)$8),)6$Q,$%.+-)#(.C$8#&"1*$&0$#)<.-)'$8),)*$.#)$&0-),$+<"*-)#)'$-&8)-/)#$.,'$-#.,*+#(%)'$(,-&
P>?$0#&3$&,)$1#&3&-)#6$=/)*)$+<"*-)#*$&0$8),)*$+&,-#&<<)'$%4$.$*(,8<)$1#&3&-)#$.#)$+.<<)'
&1)#&,*6$$
=/)$-/#))$8),)*$K()(*+'()(,'.,' ()(-L$-/.-$+&')$0&#$-/#))$'(8)*-(9)$),:43)*$(,9&<9)'$(,
-/)$%#).@'&2,$&0$.#.%(,&*)$.#)$+<"*-)#)'$-&8)-/)#$(,$2/.-$(*$@,&2,$.*$-/)$.#.%(,&*)$&1)#&,6A
=/)*)$-/#))$1#&-)(,*$.#)$')1),'),-$&,$(,(-(.-(&,$&0$-#.,*+#(1-(&,$0#&3$.$*(,8<)$1#&3&-)#C$R*,-6
=#.,*+#(1-(&,$&0$-/)*)$-/#))$8),)*$#)G"(#)*$-/)$*(3"<-.,)&"*$1#)*),+)$&0$-/)$7>?$-)31<.-)
K1#&3&-)#$.,'$&1)#&,LC$P>?$1&<43)#.*)C$.$7>?$%(,'(,8$1#&-)(,$+.<<)'$()(. .,'$.#.%(,&*)6
()(. %(,'*$-&$-/)$7>?$.-$-/)$%(,'(,8$*(-)$0&#$-/)$P>?$1&<43)#.*)$K-/)$%)8(,,(,8$&0$-/)$
.#.%(,&*)$&1)#&,L6$S/),$.#.%(,&*)$(*$1#)*),-$(,$-/)$),9(#&,3),-C$%.+-)#(.$-.@)$(-$"16$!,+)
(,*(')C$-/)$.#.%(,&*)$(,-)#.+-*$'(#)+-<4$2(-/$()(. 2/(+/$(*$%&",'$-&$-/)$7>?6$=/)$(,-)#.+-(&,
+."*)*$()(. -&$+/.,8)$(-*$*/.1)$2/(+/$(,$-"#,$1#&3&-)*$K.+-".<<4$/)<1*L$-/)$%(,'(,8$&0$P>?
1&<43)#.*)$.,'$-/)$-/#))$8),)*$()(*C$, .,'$-C$.#)$-#.,*+#(%)'6$=/#))$),:43)*$.#)$1#&'"+)'C
-/)4$%#).@$'&2,$.#.%(,&*)C$.,'$)9),-".<<4$-/)$.#.%(,&*)$#",*$&"-6$Q,$-/)$.%*),+)$&0$.#.%(,&*)
-/)$()(T$#)-"#,*$-&$(-*$&#(8(,.<$*/.1)$.,'$-#.,*+#(1-(&,$(*$*/"-$&006
=/)$7>?$+&')$&0$-/)$1JU!$1<.*3('$/.*$%)),$),8(,))#)'$-&$(,+&#1&#.-)$.*1)+-*$&0$-/)
.#.%(,&*)$&1)#&,6$V&-/$-/)$1#&3&-)#$KR*,-L$.,'$-/)$()(T$8),)$.#)$1#)*),-6$N&2)9)#C$-/)
8),)*$2/(+/$+&')$0&#$.#.%(,&*)$+.-.%&<(*3C$()(*+','.,' -C$/.9)$%)),$#)1<.+)'$%4$-/)$*(,8<)
8),)$2/(+/$+&')*$0&#$JMR6$=/)#)0&#)C$(,$-/)$1#)*),+)$&0$.#.%(,&*)C$()(. 1#&-)(,$1#&3&-)*
-/)$%(,'(,8$&0$P>?$1&<43)#.*)$.,'$JMR$(*$1#&'"+)'6$T)<<*$0<"&#)*+)$%#(<<(.,-$8#)),$.*$-/)4
1#&'"+)$3&#)$.,'$3&#)$JMR6$Q,$-/)$.%*),+)$&0$.#.%(,&*)C$()(. ,&$<&,8)#$0.+(<(-.-)*$-/)
%(,'(,8$&0$P>?$1&<43)#.*)$.,'$-/)$JMR$8),)$(*$,&-$-#.,*+#(%)'6$S/),$JMR$(*$,&-$3.')C
%.+-)#(.$+&<&,()*$2(<<$.11).#$-&$/.9)$.$2(<'W-41)$K,.-"#.<L$1/),&-41)X&0$2/(-)$+&<&,()*
2(-/$,&$0<"&#)*+),+)6$$
=/(*$(*$.,$)I+)<<),-$)I.31<)$&0$-/)$+),-#.<$3&<)+"<.#$0#.3)2&#@$&0$%(&<&84$(,$.+-(&,H$$
7>?P>?RP!=FQ>=P?Q=6

60

The Arabinose Operon

araC

araB

araA

araD

araA

araD

araA

araD

arabinose

P BAD

araB
araC
RNA Pol

RNA Pol

araB
araC

m RNA
P BAD

araB

RNA Pol

araD

araC

Expression of Green Fluorescent Protein

araC

GFP

arabinose

P BAD

GFP
araC
RNA Pol

RNA Pol

GFP
araC

m RNA

GFP
araC

61

RNA Pol

Appendix E References
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99:(;!<=>?"((
#$%$&$%'()*+,-$.'(A3B&%2C+3.(5*4(04$%.5*46$02*%(*5(!-)#*+'"(D%()EF(G-*%2%,H(F(I4$B02B$-(F884*$B&
;J1"()"(K"(L-*M34?'(M*-"(!"(DNO(I43..'(PQ5*41(;!<=:?"
/B&-325'(N*R340'A7*(8*.202M3-S(43,+-$031(.S.036.'((&( $%1(0(+'(D%(!"#$%&'#$'()#*+')$%1 1(+0*2%++('
G3--+-$4($%1(K*-3B+-$4(T2*-*,S'(E321&$410"(F/K(I43..'(U$.&2%,0*%'()"G"(;!<<V?"

I$4$52-6(2.($(04$136$4W(*5(F6342B$%(E$02*%$-(G$%(G*"
X3--YP(2.($(04$136$4W(*5(Z4$50([**1.'(D%B"

62